Delivery system

ABSTRACT

Provided herein is a delivery system, including: (a) an optical sensor configured to detect data to create a map of a patient bodily surface; and (b) a dispenser operatively associated with the optical sensor and configured to deliver compositions (optionally including cells) to the patient bodily surface based upon the data or map. Methods of forming a tissue on a patient bodily surface of a patient in need thereof are also provided, as are methods, systems and computer program products useful for processing patient bodily surface data.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/158,808, filed Oct. 12, 2018 which claims priority under 35 U.S.C. §120 as a divisional application of U.S. patent application Ser. No.14/019,714, filed Sep. 6, 2013, which is a continuation of PCTApplication No. PCT/US2012/027731, filed Mar. 5, 2012, which in turnclaims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication No. 61/450,021, filed Mar. 7, 2011, and U.S. ProvisionalPatent Application No. 61/507,416, filed Jul. 13, 2011, the contents ofeach of which is incorporated by reference herein in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This work was supported by grant W81XWH-08-2-0032 from the Armed ForcesInstitute for Regenerative Medicine. The U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention concerns the in situ delivery of viable cells ontoa subject.

BACKGROUND OF THE INVENTION

In the United States, the mortality rate for burns is approximately 4.9%and increases dramatically with increasing total body surface area(TBSA) burned. The gold standard of treatment is the split-thicknessautograft, but this technique requires injuring one or more sites ofundamaged skin. Other treatment techniques include silver sulfadiazine,INTEGRA® (Johnson and Johnson, Hamburg, Germany; Integra Life SciencesCorporation, NJ), BIOBRANE® (Dow Hickam/Bertek Pharmaceuticals, SugarLand, Tex.), TRANSCYTE® (Advanced Tissue Sciences, Inc., La Jolla,Calif.), and allogeneic cells.

Large-scale manufacturing processes necessitate production of standardsizes of skin substitutes, but these standard-sized products cannotadequately cover irregular wounds. In addition, nearly all of thesetechniques require multiple surgical procedures, and are not ideal forlarge body surface area burns.

Allogeneic cell therapy can eliminate the need for autologous cellculture. Current delivery techniques include spraying cells onto thepatient or seeding a scaffold with cells before implantation. Cellspraying has been used to treat burns with autologous fibroblasts andkeratinocytes, but the delivery precision of current spraying technologyis low.

The ideal skin substitute possesses the following qualities: (1) itadheres intimately to the wound bed, especially for irregular surfaces;(2) it provides a non-antigenic microbial barrier; (3) it participatesin normal host repair mechanisms; (4) it maintains elasticity andlong-term durability; (5) it displays long-term mechanical and cosmeticfunction comparable to split-thickness autografts; (6) it requires asingle surgical procedure; (7) it is inexpensive; (8) it has anindefinite shelf life; and (9) it has minimal storage requirements.

New treatments are needed that better address the needs of burn woundpatients, as well as patient having other wounds and tissue injury ordisease.

SUMMARY OF THE INVENTION

Provided herein is a delivery system including a control moduleconfigured to generate a map of a patient bodily surface based onpatient bodily surface data captured by a scanning system and togenerate a command to operate a dispensing system to deliver cellsand/or compositions to the patient bodily surface based on the map. Insome embodiments, the delivery system also includes a database includingscanning system parameters and/or dispensing system parameters storedtherein. In some embodiments, the delivery system further includes aninterface module configured to convert the patient bodily surface datainto a format suitable for use by the control module to generate the mapbased on the scanning system parameters and/or to format the command tooperate the dispensing system to deliver the cells and/or compositionsbased on the dispensing system parameters.

Also provided is a delivery system, including: (a) an optical sensorconfigured to detect data used to create a map of a patient bodilysurface; and (b) a dispensing system operatively associated with theoptical sensor and configured to deliver cells and/or compositions tothe patient bodily surface based upon the map. In some embodiments, thesensor and dispensing system (or a portion thereof) can be associatedwith one another by connection of each to a common support or frame, towhich may also be connected a subject support (e.g., a bed) to place asubject in a position for scanning of the subject's patient bodilysurface. In some embodiments, the optical sensor may be removable and/orhand-held. In some embodiments, the optical sensor includes athree-dimensional scanner. In some embodiments, the optical sensorincludes an infrared detector. In some embodiments, the optical sensoris a laser scanner.

In some embodiments, the delivery system further includes: (c) athree-dimensional plotter operatively connected with the optical sensor;and (d) a controller operatively connected with the dispensing system.

In some embodiments, the dispensing system includes one or morecartridges loaded with a composition (e.g., a composition includingcells, support compounds, growth factors, combinations thereof, etc.).In some embodiments, the cartridge includes and/or is in fluidcommunication with a plurality of printheads. In some embodiments, theprintheads include nozzles configured for delivery of cells and/orcompositions.

Methods of forming a tissue on a patient bodily surface of a patient inneed thereof are also provided, including: (a) scanning the patientbodily surface to obtain the three dimensional coordinates thereof; andthen (b) dispensing viable cells on the patient bodily surface of thepatient based upon the coordinates to thereby form the tissue. In someembodiments, the dispensing step is performed two or more times insequence to make a tissue having multiple layers.

Also provided are methods of processing patient bodily surface dataobtained from a three dimensional optical detector to provide a path toa dispensing system operatively associated to the optical detector, themethods including: interpreting the patient bodily surface data from theoptical detector to form a model of the patient bodily surface;transforming the model into a negative mold of the patient bodilysurface, which mold is split into a plurality of Z-axis layers, whichlayers correspond to one or more tissue layers; and overlaying each ofthe tissue layers with a series of lines which cover the patient bodilysurface, wherein the lines provide a path for the dispensing system. Insome embodiments, the methods further include the step of obtaining thepatient bodily surface data by scanning with a three-dimensional opticalsensor. In some embodiments, the patient bodily surface data is woundsurface data (e.g., skin wound surface data).

Further provided are systems for processing data of a patient bodilysurface obtained from a three dimensional optical detector to provide apath to a dispensing system operatively associated to an opticaldetector, the system including: means for interpreting the patientbodily surface data from the optical detector to form a model of thepatient bodily surface; means for transforming the model into a negativemold of the patient bodily surface, which mold is split into a pluralityof Z-axis layers, which layers correspond to one or more tissue layers;and means for overlaying each of the tissue layers with a series oflines which cover the patient bodily surface, wherein the lines providea path for the dispensing system. Some embodiments further include meansfor obtaining the patient bodily surface data. In some embodiments, thepatient bodily surface data is wound surface data (e.g., skin woundsurface data).

Computer program products are also provided for processing data of apatient bodily surface obtained from a three dimensional opticaldetector to provide a path to a dispensing system operatively associatedto the optical detector, the computer program product including acomputer readable medium having computer readable program code embodiedtherein, the computer readable program code including: computer readableprogram code which interprets the patient bodily surface data from theoptical detector to form a model of the patient bodily surface; computerreadable program code which transforms the model into a negative mold ofthe patient bodily surface, which mold is split into a plurality ofZ-axis layers, which layers correspond to one or more tissue layers; andcomputer readable program code which overlays each of the tissue layerswith a series of lines which cover the patient bodily surface, whereinthe lines provide a path for the dispensing system. In some embodiments,the patient bodily surface data is wound surface data (e.g., skin woundsurface data).

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention and,together with the description, serve to explain principles of theinvention.

FIG. 1 is a perspective view of a delivery system (5) according to someembodiments of the present invention having a printhead support (10).This portable embodiment has wheels (24), and is positioned over asubject lying on a table (30) having a bed (35). The printhead supportis operatively connected to a manipulator (20) having members (23, 22,21) configured to allow the printhead support (10) to be moveable aboutthe Z axis (member 23), the X axis (member 22), and the Y axis (member21). A subject may lie on a table (30) having a bed (35). The system (5)may have one or more locks (28) to attach to the table (30).

FIG. 2 is a bottom view of a printhead support (10) according to someembodiments having an optical sensor (11) and a plurality of printheads(12).

FIG. 3 is an alternative embodiment of a delivery system (5) having anattached computer (40), positioned over a subject lying on a table (30)having a bed (35). A cover (60) is provided above the printhead support(10) covering a portion (members 22 and 21, and the top of member 23) ofthe manipulator (20).

FIG. 4 is an alternative embodiment of a delivery system (5) having anattached light (50), positioned over a subject lying on a table (30).

FIG. 5 is an alternative embodiment of a delivery system (5) having anattached computer (40) and a cover (60) covering the manipulator and theprinthead support, positioned over a subject lying on a table (30).

FIG. 6 is an alternative embodiment of a delivery system (5) having anattached computer (40) and a cover (60) over the manipulator (20) andprinthead support (10). The system (5) is attached to a table (30)having a bed (35).

FIG. 7 is a cutout view of a printer support (10) having a camera (11),infrared sensors (15), and a plurality of printer cartridges (14)attached thereto.

FIG. 8 is an exploded view of a printer cartridge (14).

FIG. 9 is a graph of experimental results demonstrating that skin repairusing bioprinting shows significant difference in wound size between 1and 4 weeks after injury (p<0.05).

FIG. 10 is an alternative embodiment of a delivery system (5) having arobotic arm type manipulator (20) for movement about the XYZ axis, andhaving a cover (60) over the printhead support (not shown). The deliverysystem (5) is attached to a table (30) having a bed (35).

FIG. 11 is a representative embodiment of components of a dispensingsystem. Separate reservoirs are provided for carrier/supportcompositions (310) and crosslinker (312). Cartridges (14) containingcells and/or compositions are loaded into a motorized storage unit (360)that connects a cartridge (14) into fluid communication with thecarrier/support composition. The cells and/or compositions are drawninto a mixing chamber (340) in which the cells and/or compositions canbe mixed with the carrier/support compositions prior to dispensing to atarget site. One or more pumps (350) are provided to drawcarrier/support compositions and/or crosslinker into one or moreprintheads (not shown) for dispensing.

FIG. 12 is a schematic diagram of an embodiment of a dispensing system(201). The dispensing system includes a carrier reservoir (310) in fluidcommunication with a cartridge (14), which, in turn, is in fluidcommunication with a mixing chamber (340) in which the carrier and thecartridge contents can be mixed prior to delivery through a printhead(12). A separate crosslinker reservoir (312) is provided that is influid communication with a separate printhead (12), such that thecrosslinker may be delivered contemporaneously with the mixed carrierand cartridge contents.

FIG. 13 is a diagram of a scanning system (101) communicating with aninterpreter (110).

FIG. 14 is a diagram of a dispensing system (201) communicating with aninterpreter (210).

FIG. 15 is a diagram of scanning system (101) and dispensing system(201) communicating with interpreters (110, 210).

FIG. 16 presents data on wound size, contracture andre-epithelialization of untreated, matrix only, allogeneic andautologous treatments over an 8-week period.

FIG. 17 shows H&E staining of skin biopsies taken from the center of thewound at week 2, 4 and 6, of untreated, matrix only, allogeneic andautologous treatments, to detect the formation of the epidermal anddermal layers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided herein and further described below are systems, compositions,devices and methods useful for the delivery of cells and tissues onto asubject in need thereof. In some embodiments, a cartridge based celldelivery system including a dispensing system is provided, whichdispensing system is operatively associated with an optical sensor.

Aspects of the present invention are described herein with reference tothe accompanying drawings and examples, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likenumbers refer to like elements throughout. In the figures, the thicknessof certain lines, layers, components, elements or features may beexaggerated for clarity.

The disclosures of all United States patent references cited herein arehereby incorporated by reference to the extent they are consistent withthe disclosure set forth herein. As used herein in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. Furthermore, the terms “about” and“approximately” as used herein when referring to a measurable value suchas an amount of a compound, dose, time, temperature, and the like, ismeant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% ofthe specified amount. Also, as used herein, “and/or” or “/” refers toand encompasses any and all possible combinations of one or more of theassociated listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the relevant art. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expressly sodefined herein. Well-known functions or constructions may not bedescribed in detail for brevity and/or clarity.

The present invention is described herein, in part, with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems) and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer program instructions. These computer program instructions maybe provided to a processor of a general purpose computer, specialpurpose computer, or other programmable data processing apparatus toproduce a machine, such that the instructions, which execute via theprocessor of the computer or other programmable data processingapparatus, create means for implementing the functions/acts specified inthe flowchart and/or block diagram block or blocks.

It will be understood that when an element is referred to as being “on,”“attached” to, “connected” to, “coupled” with, “contacting,” etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on,” “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

Spatially relative terms, such as “under,” “below,” “lower,” “over,”“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of “over” and “under.” The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly,” “downwardly,” “vertical,” “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

It will be understood that, although the terms “first,” “second,” etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element discussed below couldalso be termed a “second” element without departing from the teachingsof the present invention. The sequence of operations (or steps) is notlimited to the order presented in the claims or figures unlessspecifically indicated otherwise.

As illustrated in FIG. 1, in some embodiments a delivery system (5) isprovided which includes a printhead support (10) thereon. In someembodiments, the delivery system may be provided on wheels (24) forportability. The delivery system (5) may be positioned over a subjectlying on a table (30) having a bed (35) when in use. In someembodiments, a locking mechanism (28) may be provided to lock thedelivery system (5) in place relative to the table (30) and/or bed (35).The printhead support (10) is operatively connected to a manipulator(20) having members (23, 22, 21) configured to allow the printheadsupport (10) to be moveable about the Z axis (member 23), the X axis(member 22), and the Y axis (member 21).

As illustrated in FIG. 2, in some embodiments the printhead support (10)includes an optical sensor (11) and a plurality of printheads (12). Inother embodiments, the printhead support (10) includes a plurality ofprintheads (12), but the optical sensor (11) is provided on a separatesupport and/or can be (not shown).

Some alternative embodiments of the delivery system (5) are illustratedin FIGS. 3-6. FIG. 3 illustrates an embodiment of the delivery systemhaving an attached computer (40), with the printer support (10)positioned over a subject lying on a table (30). FIG. 4 illustratesanother embodiment, and includes an attached light (50), positioned overa subject lying on a table (30). FIG. 5 illustrates an embodiment havingan attached computer (40) and a cover (60) over the printhead support(10), positioned over a subject lying on a table (30). FIG. 6illustrates an embodiment of a delivery system (5) having an attachedcomputer (40) and a cover (60) over the manipulator (20) and theprinthead support (10). The delivery system (5) is attached to a table(30).

FIG. 7 illustrates an embodiment of a printhead support (10) having acamera (11), infrared sensors (15), and a plurality of cartridges (14).FIG. 8 provides an exploded view of one embodiment of a cartridge.

FIG. 10 illustrates an embodiment of a delivery system (5) having arobotic arm type manipulator (20) for movement about the XYZ axis, andhaving a cover (60) over the printhead support (not shown). The deliverysystem (5) is attached to a table (30) having a bed (35).

FIG. 11 illustrates an embodiment of components of a dispensing system.Separate reservoirs are provided for carrier/support compositions (310)and crosslinker (312). A storage unit (360), which may be motorized, isprovided in which a plurality of cartridges (14) containing cells and/orcompositions may be loaded. The cartridges (14) can be locked into fluidcommunication with the carrier/support composition according to someembodiments, using any appropriate locking mechanism known in the art. Afirst and second pump (350) are provided to draw carrier/supportcompositions and crosslinker, respectively, into one or more printheads(not shown) for dispensing. The cells and/or compositions in thecartridge (14) are drawn by the first pump (350) into a mixing chamber(340) in which the cells and/or compositions can be mixed with thecarrier/support compositions held in the reservoir (310) prior todispensing to the target site. In some embodiments, the cells and/orcompositions may be connected in a manner to allow recirculating flow,e.g., to continually mix the cell solution with carrier (not shown).

FIG. 12 provides a schematic diagram of an embodiment of a dispensingsystem (201) having a carrier reservoir (310) connected to a cartridge(14), which, in turn, is connected to a mixing chamber (340) in whichthe carrier and the cartridge contents can be mixed prior to deliverythrough a printhead (12). A separate crosslinker reservoir (312) isprovided that may be connected to a separate printhead (12), such thatthe crosslinker may be delivered contemporaneously with the mixedcarrier and cartridge contents. In some embodiments, a pump (not shown)may be provided to draw carrier and cartridge contents into the mixingchamber (340), where it is mixed. The mixed contents are then drawn intothe printhead (12) and delivered to the target site.

Referring again to FIG. 11, in some embodiments, a cartridge (14) has atleast one input port (140) and at least one output port (141). Aseparate reservoir (310) may have connectors (315) configured to fitinto the input port (140) of a cartridge (14).

A locking mechanism may attach one or more of the connectors (315) to acartridge (14) and aid in preventing the connections from breakingloose. In some embodiments, when moving to the next cartridge (14), thedispensing system releases the locked connections and spins the storageunit (360) to the next selected cartridge (14). Any suitable lockingmechanisms known in the art may be used. For example, a threaded lock,pin-type lock, etc. See, e.g., U.S. Pat. No. 1,662,482 to Ward and U.S.Pat. No. 4,119,237 to Greenwald et al.

Each of the dispensing system components can be mounted anywhere on thesystem, depending on the size of the reservoirs (310 and 312) andstorage unit (360). In some embodiments, one or more of the dispensingsystem components are mounted onto the manipulator (20). In someembodiments, larger components (e.g., reservoirs, pumps, storage unit)may be mounted to the frame, whereas smaller components (e.g., mixingchamber) may be mounted onto the manipulator (20).

An aspect of the present invention is the use of an apparatus asdescribed herein in a method for generating tissue such as for treatinga wound (e.g., burns, abrasions, lacerations, incisions, pressure sores,puncture wounds, penetration wounds, gunshot wounds, crushing injuries,etc.) in a subject in need thereof, in which cells and/or compositionsare applied thereto in an amount effective to generate the tissue ortreat the wound.

Accordingly, in some embodiments the apparatus may be used to dispensecells and/or compositions onto a patient bodily surface. “Bodilysurface” as used herein refers to the exposed tissues of one or morebody parts or a portion thereof (for example, skin or other tissuesexposed upon injury thereof) which are detectable by the opticaldetector as described herein, and include, but are not limited to, thatof the torso, chest, stomach, shoulder, back, buttocks, neck, head,face, cheek, lips, eyebrow, scalp, ear, chin, arm, elbow, hand, finger,leg, knee, foot, toe, etc. The surface may in some embodiments have asurface area of from 0.1, 0.5, 1, 5, 10, 20, or 50 cm², to 100, 120,150, 200, 250, 500, 750 or 1000 cm².

Examples of wounds that can be treated with the present inventioninclude burn wounds. Burn wounds are tissue injuries that can resultfrom heat, chemicals, sunlight, electricity, radiation, etc. Burnscaused by heat, or thermal burns, are the most common. Chemical burnsresemble thermal burns. Though burn wounds tend to occur most often onthe skin, other body structures may be affected. For example, a severeburn may penetrate down to the fat, muscle or bone. In some embodiments,cells corresponding to one or more of these tissues may be deliveredonto the wound site, e.g., in a layer-by-layer application that mimicsthe natural tissues, as needed or desired.

Wounds may be characterized by the depth of injury as known in the art.For example, the degree of a burn is characterized as first, second orthird depending on the depth of the tissues injured. In a first-degreeburn, only the top layer of skin (the epidermis) is damaged. Insecond-degree burns, the middle layer of skin (the dermis) is damaged.Finally, in a third-degree burn, the most severe type, the damage isdeep enough to affect the inner (fat) layer of the skin. Similarly,pressure sores of the skin are characterized as stage I (red, unbrokenskin, erythema does not fade with release of pressure), stage II(disrupted epidermis, often with invasion into the dermis), stage III(injury of the dermis), and stage IV (subcutaneous tissue is exposed).

In pressure sore wounds, pressure-induced constriction of localcapillaries results in ischemia in the affected skin. Similarly, a burnwound is ischemic due to associated capillary thrombosis. A diabeticulcer is another example of a poorly perfused wound. For these types ofwounds, where blood is not readily available to aid in the normal courseof wound healing, in some embodiments dead and/or injured tissue isremoved (debridement) prior to application of the cells and/orcompositions as provided herein.

In some embodiments, compositions may include an antimicrobial agent todecrease the risk of infection. In some embodiments, compositions mayinclude analgesics or anesthetics for pain relief, surfactants,anti-inflammatory agents, etc. See, e.g., U.S. Pat. No. 6,562,326 toMiller. Methods of attenuating swelling, such as treatment with cold(e.g., cool water, ice, etc.) and elevation of the affected area, mayalso be used.

According to some embodiments, the device may be used for both openwounds and closed wounds. In the case of closed wounds, the scannerand/or dispensing device may be allowed access to the wound site throughsurgical means, inclusive of endoscopic procedures.

In some embodiments, reapplication of the cells and/or compositions maybe performed as needed. Cleansing to remove bacteria and debridement toremove necrotic debris may also be warranted during the course oftreatment. Application of a moisturizing cream or ointment may be usedto soften wound eschar in order to assist in debridement.

A. Dispenser

In some embodiments, cells, proteins, support materials, combinationsthereof, etc., are delivered with a printer and/or other dispenser ordispensing system. “Dispenser” or “dispensing system” as used herein isa device that functions to deliver cells and/or compositions to a targetsite. In some embodiments, the cells and/or compositions in thedispensing system are delivered through a printhead or nozzle placedabove a target site or in following a predetermined path across a targetsite.

“Printing” as used herein refers to the delivery of droplets of cellsand/or compositions with small volumes, e.g., from 0.5 to 500microLiters, or 5 to 100 microLiters, or from 10 to 75 microLiters perdroplet. In some embodiments, droplets have a volume ranging from 0.5 to500 picoLiters, or 5 to 100 picoLiters, or from 10 to 75 picoLiters perdroplet. Printing may be performed by, e.g., using standard printerswith print heads that are modified as described herein.

In some embodiments, printing can provide a precise delivery of cellsand/or compositions to a resolution of approximately 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 200 μm. Printing can also deliver specificcells to specific target sites using a layer-by-layer fabrication. Suchlayer-by-layer fabrication may in some embodiments be performed in situon a subject in need thereof, and involve multiple cell types arrangedwith precision. This is in contrast to cell seeding or sprayingtechniques, in which cells are randomly applied over a large area.

In some embodiments, cells and/or compositions are delivered to targetsites using other dispensers such as a spray or stream. Such deliveryallows continuous delivery of cells and/or compositions to a targetsite, which may serve to reduce the time needed for the application ofthe cells and/or compositions to the target site. See also U.S. Pat. No.6,986,739 to Warren et al.

In some embodiments of stream dispensing, delivery pumps are activatedwhen the printhead is over a target site and can deliver cells and/orcompositions in lines of the same or varying widths across the site, asdesired. For example, lines between 0.001 and 10 cm, or 0.01 and 1 cm,or 0.1 and 0.5 cm may be provided according to some embodiments.

In some embodiments, the dispenser may include both a printer andanother dispensing system such as a spray or stream. In such a system,the spray or stream may be used for faster but less precise delivery,while the printer may be used for slower but more precise delivery ofcells and/or compositions to the target site.

As used herein, the “printhead” is the portion of a printer or otherdispensing device that applies droplets, sprays or streams of cellsand/or compositions onto the target site. In some embodiments, theprinthead includes one or more nozzles attached thereto, through whichthe cells and/or compositions are passed from the dispensing system to atarget site.

A “cartridge” as used herein refers to a vessel or reservoir in whichcells and/or compositions may be held, and which is in fluidcommunication with one or more nozzles and/or one or more printheads.The cartridge may include the reservoir and printhead in a single unit,as in a traditional inkjet cartridge, or the reservoir and nozzle may bein separate units but connected such that they are in fluidcommunication (e.g., through the use of a connector such as tubing). Insome embodiments, cartridges may hold from 0.5 mL to 25 mL, or from 1 mLto 15 mL, or from 5 mL to 10 mL in volume.

In some embodiments, one or more cell types and/or compositions areloaded into an individual cartridge. “Compositions” may include cells,carriers (e.g., hydrogels), support materials, crosslinkers,macromolecules such as proteins, cytokines, growth factors, etc., or anycombination thereof. Compositions may also include oxygen generatingbiomaterials. See, e.g., PCT Publication WO 2008/124126.

In some embodiments, the compositions may be provided in a largercontainer (e.g., reservoir) than that cartridge. In some embodiments,the container may hold from 5 mL to 5 L, or from 10 mL to 1 L, or from15 mL to 500 mL in volume of the composition (e.g., carrier, supportmaterials, crosslinkers, etc.).

In some embodiments, each cartridge may be configured to connect tomultiple nozzles and/or printheads, in contrast to standard inkjetprinting in which one printhead is connected to one cartridge. Thisallows arbitrary printhead configurations that can conform to the needsof the treatment. It also increases the throughput of the system andprovides a rapid method of sterilization by attaching a cartridge ofcleaning fluid to the printheads.

In some embodiments, the printheads contain pressure-based nozzles. Apressure-based delivery system according to some embodiments allows theprinter or other dispenser to remain a safe distance above the patientand to accommodate a variety of body types. As used herein, a“pressure-based” delivery system uses three components: the pressuresource, material reservoirs, and delivery mechanism.

In some embodiments, the delivery mechanism is a series of voltageoperated inkjet valves. The pressure source is operatively connected tothe reservoir, which is in fluid communication with the deliverymechanism. In some embodiments, a gas (e.g., air, air plus 5% CO₂, etc.)is pumped into empty space in the reservoir by the pressure source,which in turn drives the material in the reservoir (cells and/orcompositions) into the delivery mechanism.

In some embodiments, the printhead is equipped with a DC solenoid inkjetvalve. In some embodiments, one or more, or several, reservoirs forloading cells are connected to the inkjet valve. In some embodiments,the cells and/or compositions may be supplied from the reservoirs to thevalve or nozzle by air pressure.

In some embodiments, the dispenser includes a two-dimensional (X-Y) orthree-dimensional (X-Y-Z) plotter (e.g., driven by step motors). In someembodiments, the printhead may be mounted on an X-Y-Z plotter to allowprecise deposition of cells onto a target site. Positioning of the XYZplotter under the printhead may be controlled via a controller. In someembodiments, the controller acquires the positioning information fromsoftware loaded on a computer. In some embodiments, the softwareconverts the image of the target to a four-byte protocol, which is usedto activate specific inkjet valves and coordinate the X-Y-Z position.

A pressure-based system may be preferable in some embodiments ascompared to inkjet cartridges in the context of in situ printing becauseit separates the reservoir and delivery mechanism. A traditional inkjetcartridge includes the reservoir and delivery mechanism in a singleunit. If either the reservoir or delivery mechanism fails, then theentire unit fails. Furthermore, inkjet cartridges must be filled andsealed prior to printing. If the inkjet cartridge is filled with cells,failure of the cartridge means the loss of all cells contained in thatcartridge. Embodiments of the delivery systems described herein solveboth of these issues. The reservoir and delivery mechanism can bereplaced individually in the case of failure of either component.Material is only pumped to the delivery mechanism when it is needed, sofailure of the delivery mechanism should not result in the loss of allmaterial in the reservoir.

In addition, in some embodiments the pressure-based system can provide amethod of detecting and clearing clogged valves. By placing a pressuresensor at the end of the valve, the delivery pressure can be compared tothe applied pressure. If the difference between the two pressures islarger than a certain threshold, then the valve is clogged. Redirectingthe output of the valve to a waste reservoir and applying a large burstof pressure may be used to try and clear the valve. If the clearingprocess fails, then the delivery system will detect this and cancontinue printing without using that valve.

In some embodiments, the dispenser is driven by a pressure provided byinternal pumps. In some embodiments, the pumps draw cells and/orcompositions from one or more cartridges into a chamber. The chamber maybe configured to allow mixing of cells and/or compositions held inseparate cartridges prior to dispensing to the target site. In someembodiments, there is a pump operatively associated with the printheadthat draws the mixed material into the printhead for dispensing.

In some embodiments, pumps may be provided to draw compositions throughthe dispensing system or a portion thereof through negative pressureand/or positive pressure. In some embodiments, the pump is a variablespeed pump designed to have a flow rate in the dispenser between 0.01mL/second and 1 mL/sec or between 0.03 mL/sec and 0.07 mL/sec; a flowrate between 0.1 mL/sec and 1 mL/sec or between 0.25 mL/sec and 0.65mL/sec; and/or a flow rate between 0.5 mL/sec and 0.45 mL/sec. In someembodiments, the pump has a variable flow rate inclusive of rates about0.05 mL/second; about 0.45 mL/sec; and/or about 0.25 mL/sec (e.g., nomore than 10, 15 or 20% above or below these values).

In some embodiments, a system of pumps draws gel out of the reservoir,through the cartridge, into the mixing chamber, and then into theprinthead. In some embodiments, this system is closed and does notrequire an outside pressure source to move material into the printhead.In some embodiments, a continuous line is provided, and pumps areconfigured to supply negative and/or positive pressure to move cellsand/or compositions through the line without the need for an openpressure source such as compressed gas, thus providing a closed systemin which the cells can be maintained in a sterile environment.

In some embodiments, at least a portion of the dispenser components arehoused in a container configured to maintain a temperature of about 37degrees Celsius. In some embodiments, at least a portion of thedispenser components are kept at room temperature. In some embodiments,at least a portion of the dispenser components are kept at a temperaturelower than room temperature (e.g., about 4 degrees Celsius). In someembodiments, at least a portion of the dispenser are housed in acontainer configured to vary the temperature as desired (e.g., fromabout 4 degrees Celsius to room temperature, from room temperature toabout 37 degrees Celsius, or from about 4 degrees Celsius to about 37degrees Celsius).

Cells may be printed in the form of a composition that contains acarrier. The cells may be provided in the form of a suspension,solution, or any suitable form. The carrier may be a solid or a liquid,or both (e.g., a gel). In some embodiments, the cells are provided as asuspension in the carrier to reduce clumping of the cells. Supportcompounds, growth factors, etc., may be included in compositions havingcells and/or may be included in compositions without cells (but mayinclude a suitable carrier), as desired.

Suitable gels include, but are not limited to, agars, collagen, fibrin,hyaluronic acid, including hydrogels thereof, etc. Besides gels, othersupport compounds may also be utilized in the present invention.Extracellular matrix analogs, for example, may be combined with supportgels to optimize or functionalize the gel. One or more growth factorsmay also be included. In some embodiments a temperative sensitive gelmay be used. Examples of temperature sensitive gels includethermaosensitive hydrogels and thermosensitive polymer gels (e.g., apoloxamer such as Pluronic® F-127 (BASF corporation, Mont Olive, N.J.)).See also U.S. Pat. Nos. 6,201,065, 6,482,435.

Other examples of suitable liquid carriers include, but are not limitedto, water, ionic buffer solutions (e.g., phosphate buffer solution,citrate buffer solution, etc.), liquid media (e.g., modified Eagle'smedium (“MEM”), Hanks' Balanced Salts, etc.), and so forth. The use of aliquid or gel carrier in the cell composition may in some embodimentspromote adequate hydration and minimize evaporation of the cells afterprinting.

In some embodiments, cells may also be transfected (e.g., with aspecific gene) with material of interest. Useful genetic material maybe, for example, genetic sequences that are capable of reducing oreliminating an immune response in the host. For example, the expressionof cell surface antigens such as class I and class II histocompatibilityantigens can be suppressed. This would allow the transplanted cells tohave a reduced chance of rejection by the host. Cells may also betransfected with a gene encoding one or more growth factors. Accordingto some embodiments, cells may be transfected during the printingprocess. See PCT publication WO 2008/153968 to Xu et al.

The present invention includes the building of tissues by theappropriate combination of cell and support material, or two or three ormore different cell types typically found in a common tissue, preferablyalong with appropriate support compound or compounds, and optionallywith one or more appropriate growth factors. Cells, support compounds,and growth factors may be dispensed from separate nozzles or through thesame nozzle in a common composition, depending upon the particulartissue (or tissue substitute) being formed. Dispensing may besimultaneous, sequential, or any combination thereof. Some of theingredients may be dispensed in the form of a first pattern (e.g., anerodable or degradable support material), and some of the ingredientsmay be dispensed in the form of a second pattern (e.g., cells in apattern different from the support, or two different cell types in adifferent pattern). Again, the particular combination and manner ofdispensing will depend upon the particular tissue construct desired.

In embodiments in which increased delivery precision is desired, thedispenser includes a printer having thermal or piezoelectric printheadsand/or inkjet cartridges for increased delivery precision. Methods andcompositions for the inkjet printing of viable cells are known anddescribed in, for example, U.S. Pat. No. 7,051,654 to Boland et al.;Wilson et al. (2003) The Anatomical Record Part A 272A: 491-496.

In some embodiments, cells/compositions are dispensed by printing with amodified inkjet printer. Modifications may include, but are not limitedto, means to control the temperature, humidity, shear force, speed ofprinting, and firing frequency, by modifications of, e.g., the printerdriver software and/or the physical makeup of the printer. See, e.g.,Pardo et al. (2003) Langmuir 19:1462-1466; U.S. Pat. No. 7,051,654 toBoland et al. Not every modification suggested in these references willbe suitable to a given application, as will be appreciated by thoseskilled in the art. For example, in some embodiments, printers are notmodified by using new gear mount pillars with closer tolerances byadding a horizontal support, changing the transistor in the circuit toone with higher amplification, and reentering the horizontal positionencoder. Also, in some embodiments, printer software is not modified tolower the resistive voltages to avoid heating of the solutions above 37°C.

In some embodiments, printers (e.g., the commercial printers HP695C andHP550C) may be modified as follows. The printer top cover may be removedand the sensor for the cover disabled. The paper feeding mechanism maybe disabled to allow printing of cells onto solid substrates (e.g.,scaffolds). The ink absorbing pads (which are on the right side of theHP695C and HP550C printers) may be removed (e.g., to avoid the padscontaminating the bottom of the print cartridges during the printingprocess). To offer the capability of the printer to print 3D constructs,a customized Z-axis module with a controlled elevator chamber may beadded.

In some embodiments, the printer is a thermal bubble inkjet printer. Ingeneral, in a thermal bubble inkjet printer, resistors create heat inthe print head, which vaporizes ink to create a bubble. As the bubbleexpands, some of the ink is pushed out of a nozzle onto the paper. Avacuum is created when the bubble collapses, which pulls more ink intothe print head from the cartridge. In the present invention, the ink isreplaced with, e.g., cells and/or compositions of interest (e.g., cellsin a liquid carrier), and the paper is replaced with a suitablesubstrate, e.g., an agar or collagen coated substrate, or a suitablescaffold. See, e.g., U.S. Pat. No. 6,537,567 to Niklasen et al.

In other embodiments, cells are printed using a piezoelectric crystalvibration print head. In general, a piezoelectric crystal receives anelectric charge that causes it to vibrate, forcing ink out of thenozzle, and pulling more ink into the reservoir. In the presentinvention, the ink is replaced with, e.g., cells and/or compositions ofinterest. Compared with the thermal inkjet printing, the piezo-basedinkjet printing usually requires more power and higher vibrationfrequencies. Typical commercial piezo-printers use frequencies up to 30kHz and power sources ranging from 12 to 100 Watts. Therefore, in someembodiments a piezoelectric crystal vibration print head is used, with avibrating frequency of 1, 5, 10 or 15, to 20, 25, 30, or 35 or more kHz,and power sources from 5, 10, 20, 50, 100, 120, or 150, to 200, 250,300, 350, or 375 or more Watts.

In some embodiments, the printhead nozzles are each independentlybetween 0.05 and 200 μm in diameter, or between 0.5 and 100 μm indiameter, or between 10 and 70 μm, or between 20 and 60 μm in diameter.In further embodiments, the nozzles are each independently about 40 or50 μm in diameter. In still further embodiments, the nozzles are eachindependently between 0.1 or 0.5 and 2 or 3 mm. A plurality of nozzleswith the same or different diameters may be provided. A more narrownozzle may give greater precision delivery but low throughput, and viceversa. These may be provided according to cell type and/or precisiondesired. Though in some embodiments the nozzles have a circular opening,other suitable shapes may be used, e.g., oval, square, rectangle, etc.,without departing from the spirit of the invention.

In some embodiments, the printhead nozzles and/or dispensing system isconfigured to deliver compositions as drops. In some embodiments, thedrops may be 0.5, 1, 10 or 50 microliters, to 75, 100, 150, 200, 250,300, 350, 400, 450, or 500 microliters in volume.

In some embodiments, the dispensing system is configures to deliver alayer of cells/compositions at a rate of 1, 5, 7, 10, 15, 20, 25, or 30minutes, to 45, 60, 90, or 120 minutes per cell layer.

In some embodiments, the cells/compositions are formulated to provide anencapsulated form upon dispensing. The encapsulation of cells inpermeable capsules is known and described in, for example, U.S. Pat. No.6,783,964. For example, the cells may be encapsulated in a microcapsuleof from 50 or 100 μm to 1 or 2 mm in diameter that includes an internalcell-containing core of polysaccharide gum surrounded by a semipermeablemembrane; a microcapsule that includes alginate in combination withpolylysine, polyornithine, and combinations thereof. Other suitableencapsulating materials include, but are not limited to, those describedin U.S. Pat. No. 5,702,444.

“Encapsulated” cells are cells or small clusters of cells or tissue thatare surrounded by a selective membrane laminate that allows passage ofoxygen and other required metabolites, releases certain cell secretions(e.g., insulin), but limits the transport of the larger agents of thehost's immune system to prevent immune rejection. Encapsulation may beuseful for, e.g., the delivery of cells and/or tissues containingxenogeneic or allogeneic cells while reducing the risk of immunerejection in a host. This may be useful, e.g., to treat diseases due toinadequate or loss or secretory cell function, or ailments that wouldbenefit from the addition of certain secretory cells.“Microencapsulation” of cells is where one, two, three or several cellsare encapsulated. In some embodiments, each membrane encapsulates 10cells or less, preferably 5 cells or less, of at least 50, 70, 80, 90 or95% or more of the printed cells. See also U.S. Patent ApplicationPublication No. 2009/0208577 to Xu et al.

In some embodiments, two or more layers may be separately applied, withsubsequent layers applied to the top surface of previous layers. Thelayers can, in one embodiment, fuse or otherwise combine followingapplication or, alternatively, remain substantially separate and dividedfollowing application to the subject.

The thickness of a printed layer (e.g., cell layer, support layer, etc.)may generally vary depending on the desired application. For example, insome embodiments, the thickness of a layer containing cells is fromabout 2 micrometers to about 3 millimeters, and in some embodiments,from about 20 micrometers to about 100 micrometers. Further, asindicated above, support compounds, such as gels, may be used tofacilitate the survival of printed cells.

“Support compounds” which may be included in compositions may be anynaturally occurring or synthetic support compound, includingcombinations thereof, suitable for the particular tissue being printed.In general, the support compound is preferably physiologicallyacceptable or biocompatible. Suitable examples include, but are notlimited to, alginate, collagen (including collagen VI), elastin,keratin, fibronectin, proteoglycans, glycoproteins, polylactide,polyethylene glycol, polycaprolactone, polycolide, polydioxanone,polyacrylates, polysulfones, peptide sequences, proteins andderivatives, oligopeptides, gelatin, elastin, fibrin or fibrinogen,laminin, polymethacrylates, polyacetates, polyesters, polyamides,polycarbonates, polyanhydrides, polyamino acids carbohydrates,polysaccharides and modified polysaccharides, and derivatives andcopolymers thereof (see, e.g., U.S. Pat. Nos. 6,991,652 and 6,969,480)as well as inorganic materials such as glass such as bioactive glass,ceramic, silica, alumina, calcite, hydroxyapatite, calcium phosphate,bone, and combinations of all of the foregoing. The support compound maybe provided in a hydrogel, and crosslinked after delivery, if desired.

When dispensing certain types of two-dimensional or three-dimensionaltissues or portions thereof, it is sometimes desired that any subsequentcell growth is substantially limited to a predefined region. Thus, toinhibit cell growth outside of this predefined region, compounds may beprinted or otherwise applied to the print area that inhibit cell growthand thus form a boundary for the printed pattern. Some examples ofsuitable compounds for this purpose include, but are not limited to,agarose, poly(isopropyl N-polyacrylamide) gels, and so forth.

In one embodiment, for instance, this “boundary technique” may beemployed to form a multi-layered, three-dimensional tube of cells, suchas blood vessels. For example, a cell suspension may be mixed with afirst gel (“Gel A”) in one nozzle, while a second gel (“Gel B”) isloaded into another nozzle. Gel A induces cell attachment and growth,while Gel B inhibits cell growth. To form a tube, Gel A and the cellsuspension are printed in a circular pattern with a diameter and widthcorresponding to the diameter and wall thickness of the tube, e.g., fromabout 3 to about 10 millimeters in diameter and from about 0.5 to about3 millimeters in wall thickness. The inner and outer patterns are linedby Gel B defining the borders of the cell growth. For example, a syringecontaining Gel A and “CHO” cells and a syringe containing Gel B may beconnected to the nozzle. Gel B is printed first and allowed to cool forabout 1 to 5 minutes. Gel A and CHO cells are then printed on theagarose substrate. This process may be repeated for each layer.

B. Optical Detector

In some embodiments, the area or areas of interest onto which cellsand/or compositions are to be delivered is detected by an opticaldetector device to determine the two-dimensional and/orthree-dimensional map thereof. The optical detector is, in someembodiments, operatively associated with an attached cell deliverydevice, such that the cell delivery pattern may be optimized for in situdelivery of the cells and/or compositions based upon such map.

“Map” as used herein refers to the two- and/or three-dimensional surfacemeasurements, coordinates, and/or any other data that may represent thetwo- and/or three-dimensional surface of an area of interest (e.g., awound). The map may be updated by scanning at any time, and/or in realtime during delivery of cells and/or compositions.

As used herein, the “optical detector” may comprise one or moredetectors that detect light at various wavelengths, e.g., visible light,infrared, ultraviolet, combinations thereof, etc. In some embodiments,both visible light and infrared light are detected. The optical detectormay also have a depth detector to allow portability and/or account formovement of a subject during the scanning.

In some embodiments, optical reference points (e.g., dots, lines, etc.)may be placed around the wound area or in proximity thereof, which maybe used to calibrate the optical detector. This may allow the subject tobe moved without the need to re-calibrate the optical detector.

In some embodiments, an optical detector such as a camera may be used tocapture images that coincide with the surface measurements of the areaof interest. In some embodiments, an image sensor is used to collectlight reflected from an object and generate an image of the object. Amirror and lens system may be combined with the imaging device to focusthe light reflected by the object onto the image sensor. The imagesensor may be one of a charge coupled device (CCD) and a complementarymetal oxide semiconductor (CMOS), typically arranged into an area array,although the invention is not so limited. The number of sensors, eachrepresenting a pixel (short for “picture element”), determine theresolution of the image taken. A pixel is the smallest unit that makesup a digital image, and can represent the shade and/or color of aportion of an image. The output of a set of image sensors may be encodedas a set of pixels to create a digital image. The digital image may bestored in a compressed format such as in a jpeg, tiff, and/or gifformat, among others. The image may then be stored in a digital storagedevice and may be displayed on a monitor by a display application.

In some embodiments, the optical detector is a three dimensional scannersuch as that described in U.S. Pat. No. 6,856,407 to Knighton et al.(incorporated by reference herein), in which depth data for athree-dimensional object may be calculated from an intensity differenceresulting from an intensity gradient projected on the object capturingan intensity at a location on a surface in a single pixel of an imagesensing array (ISA). The intensity may be converted into a measurementof distance to the location relative to a reference point independentlyof data from other pixels of the ISA and independent of time of flightof light reflected from the location to the single pixel. A plurality ofcaptures of the intensity at the location may be compared underdifferent conditions to compensate for non-homogeneous environments orsurfaces.

In some embodiments, the optical detector is a three dimensional scanneras described in U.S. Patent Application Publication No. 2005/0237581 toKnighton et al. (incorporated by reference herein). The scanning devicemay be used to generate three dimensional representation of an area ofinterest. As used herein, three dimensional representations may be anytype of digital modeling, abstraction and/or similar techniques that mayutilize depth maps, polygon meshes, parametric solids, point clouds andsimilar data structures to create and store a three dimensionalrepresentation of the scanned object. The scanner may include a lens orset of lenses to focus light on one or more image sensing arrays (ISA).In some embodiments, the ISAs may be a charged coupled device (CCD),complementary metal oxide semiconductor (CMOS) sensor, or similarimaging array. In some embodiments, lenses may be replaced by and/orsupplemented with a reflector, light guide and/or similar article. Byvarying the focal settings, different aspects of the relief of an objectmay be brought into focus on an ISA. In some embodiments, an opticalsystem having one or more optical elements distributes a same view of atarget to a plurality of ISA's, each having a different focal rangerelative to the target.

Stereovision may also be used. Traditional stereovision methods estimateshape by establishing spatial correspondence of pixels in a pair ofstereo images. A new concept called spacetime stereo has been developed,which extends the matching of stereo images into the time domain. Byusing both spatial and temporal appearance variations, it was shown thatmatching ambiguity could be reduced and accuracy could be increased. Theshortcoming of spacetime stereo or any other stereo vision method isthat matching of stereo images is time-consuming, therefore making itdifficult to reconstruct high-resolution 3D shapes from stereo images inreal time.

Further vision based surface mapping techniques may use structuredlight, which includes various coding methods and employs varying numberof coded patterns. Unlike stereo vision methods, structured lightmethods usually use processing algorithms that are much simpler.Therefore, it becomes possible to achieve real-time performance, i.e.,measurement and reconstruction. For real-time shape measurement, thereare basically two approaches. The first approach is to use a singlepattern, typically a color pattern. The use of this approach employs acolor-encoded Moire technique for high-speed 3D surface contourretrieval. Other techniques use a rainbow 3D camera for high-speed 3Dvision. Still others use a color structured light technique forhigh-speed scans of moving objects. Because these methods use color tocode the patterns, the shape measurement result is affected to varyingdegrees by the variations of the object surface color. In general,better accuracy is obtained by using more patterns.

Another structured light approach for real-time shape measurement is theuse of multiple coded patterns with rapid switching between them so thatthey could be captured in a short period of time. This approach has beenused and develops a real-time 3D model measurement system that uses fourpatterns coded with stripe boundary codes. Some embodiments may providean acquisition speed of about 15 fps, which is sufficient for scanningslowly moving objects. However, like any other binary-coding method, thespatial resolution of these methods is relatively low because the stripewidth must be larger than one pixel. Moreover, switching the patterns byrepeatedly loading patterns to the projector may limit the switchingspeed of the patterns and therefore the speed of shape measurement.

Another example of an optical detector is found in U.S. Pat. Nos.6,788,210 and 6,438,272 (incorporated by reference herein), whichprovide a vision system for real-time and high-speed 3D shapemeasurement, with full capability of providing fast updating of the 3Dsurface maps and maps of the curves indicating the treatment areas andpositioning markings such as tick marks of the said curves. In thismanner, the sensor may serve to close the present automated debridementsystem control loop and as the means to provide for safe operation ofthe system, by for example, shutting the treatment laser beam off whenthe error between the actual position and desired position of thetreatment laser beam is more than a selected (programmed) threshold.This method is based on a rapid phase-shifting technique. This techniqueuses three phase-shifted, sinusoidal grayscale fringe patterns toprovide pixel-level resolution. The patterns are projected to the objectwith a switching speed of 240 fps. This system takes full advantage ofthe single-chip DLP technology for rapid switching of three coded fringepatterns. A color fringe pattern with its red, green, and blue channelscoded with three different patterns is created by a PC. When thispattern is sent to a single-chip DLP projector, the projector projectsthe three color channels in sequence repeatedly and rapidly. Toeliminate the effect of color, color filters on the color wheel of theprojector are removed. As a result, the projected fringe patterns areall in grayscale. A properly synchronized high-speed B/W CCD camera isused to capture the images of each color channel from which 3Dinformation of the object surface is retrieved. A color CCD camera,which is synchronized with the projector and aligned with the B/Wcamera, is also used to take 2D color pictures of the object at a framerate of 26.7 fps for texture mapping. Together with the fast 3Dreconstruction algorithm and parallel processing software,high-resolution, real-time 3D shape measurement is realized at a framerate of up to 40 fps and a resolution of 532×500 points per frame. Othersystems for 3D shape measurement known in the art can also be used inthe system and methods of the present invention, such as thosecommercially available from Blue Hill Optical Technologies, located inNorwood, Mass. or Nutfield Technology, Windham, N.H.

For the projection of the computer-generated patterns, a single-chip DLPprojector is used, which produces images based on a digital lightswitching technique. With this system, a complex facial surface has beenmapped at 40 fps (the accuracy of the system being 0.1×0.1×0.1 mm),providing an excellent speed and resolution for the present automatedlaser debridement and treatment systems and the like.

The color image is produced by projecting the red, green, and bluechannels sequentially and repeatedly at a high speed. The three colorchannels are then integrated into a full color image. To take advantageof this projection mechanism of a single-chip DLP projector, a colorpattern which is a combination of three patterns in the red, green, andblue channels is created. The projector has no color filters for amonochrome mode of operation. As a result, when the color pattern issent to the projector, it is projected as three grayscale patterns,switching rapidly from channel to channel at 240 fps. A high-speed B/Wcamera, which is synchronized with the projector, is used to capture thethree patterns rapidly for real-time 3D shape measurement. An additionalcolor camera is used to capture images for texture mapping. To obtain 3Dmaps and color information simultaneously, multi-threading programmingis used to guarantee that two cameras work independently and that thetiming of image grabbing is only determined by the external triggersignal.

For more realistic rendering of the object surface, a color texturemapping method may be used that is based on a sinusoidal phase-shiftingmethod. In this method, the three fringe patterns have a phase shift of2π/3 between neighboring patterns. Since averaging the three fringepatterns washes out the fringes, a color image can be obtained withoutfringes by setting the exposure time of the color camera to oneprojection cycle or 12.5 ms.

These systems provide the capability of rapidly projecting and capturingthree coded patterns rapidly. The employed fast three-stepphase-shifting method provides a real-time 3D reconstruction speed andhigh measurement accuracy of the order of 0.1×0.1×0.1 mm. The sinusoidalphase-shifting method that has been used extensively in opticalmetrology to measure 3D shapes of objects at various scales. In thismethod, a series of phase-shifted sinusoidal fringe patterns arerecorded, from which the phase information at every pixel is obtained.This phase information helps determine the correspondence between theimage field and the projection field. Once this correspondence isdetermined, the 3D coordinate information of the object can be retrievedbased on triangulation. A number of different sinusoidal phase-shiftingalgorithms are available. A three-step phase-shifting algorithm similarto the traditional three-step algorithm may be used, which requiresthree phase-shifted images.

In some embodiments, the optical detector accounts for movement of thesubject (e.g., breathing movements of the torso) by including a rangecamera that interprets 3D scene information from acontinuously-projected infrared structured light. In some embodiments,the optical detector includes an RGB camera and depth sensor. The depthsensor may include an infrared laser projector combined with amonochrome CMOS sensor, which captures video data in 3D under ambientlight conditions. The sensing range of the depth sensor may beadjustable, and in some embodiments the device software is capable ofautomatically calibrating the sensor based on the detected environment,accommodating for a variety of targets and body types.

C. Cells and Tissues

Any type of cell may be printed using the methods herein, including, butnot limited to, mammalian cells (including mouse, rat, dog, cat, monkeyand human cells), including somatic cells, stem cells, progenitor cellsand differentiated cells, without limitation. Stem cells have theability to replicate through numerous population doublings (e.g., atleast 60-80), in some cases essentially indefinitely, and also have theability to differentiate into multiple cell types (e.g., is pluripotentor multipotent). It is also possible for cells to be transfected with acompound of interest that results in the cells becoming immortalized(i.e., able to double more than 50 times). For example, it has beenreported that mammalian cell transfection with telomerase reversetranscriptase (hTERT) can immortalize neural progenitor cells (See U.S.Pat. No. 7,150,989 to Goldman et al.).

“Embryonic stem cell” as used herein refers to a cell that is derivedfrom the inner cell mass of a blastocyst and that is pluripotent.

“Amniotic fluid stem cell” as used herein refers to a cell, or progenyof a cell, that (a) is found in, or is collected from, mammalianamniotic fluid, mammalian chorionic villus, and/or mammalian placentaltissue, or any other suitable tissue or fluid from a mammalian donor,(b) is pluripotent; (c) has substantial proliferative potential, (d)optionally, but preferably, does not require feeder cell layers to growin vitro, and/or (e) optionally, but preferably, specifically bindsc-kit antibodies (particularly at the time of collection, as the abilityof the cells to bind c-kit antibodies may be lost over time as the cellsare grown in vitro).

“Pluripotent” as used herein refers to a cell that has completedifferentiation versatility, e.g., the capacity to grow into any of theanimal's cell types. A pluripotent cell can be self-renewing, and canremain dormant or quiescent with a tissue. Unlike a totipotent cell(e.g., a fertilized, diploid egg cell) a pluripotent cell cannot usuallyform a new blastocyst.

“Multipotent” as used herein refers to a cell that has the capacity togrow into any of a subset of the corresponding animal cell types. Unlikea pluripotent cell, a multipotent cell does not have the capacity toform all of the cell types of the corresponding animal.

Cells may be autologous (i.e., from the very subject to which they willbe applied) syngeneic (i.e., genetically identical or closely related,so as to minimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species). Syngeneic cells includethose that are autogeneic (i.e., from the subject to be treated) andisogeneic (i.e., a genetically identical but different subject, e.g.,from an identical twin). Cells may be obtained from, e.g., a donor(either living or cadaveric) or derived from an established cell strainor cell line. For example, cells may be harvested from a donor usingstandard biopsy techniques known in the art.

According to some embodiments, at least a portion of the cells areviable after they are printed. “Viable cells” includes cells that adhereto a culture dish or other substrate and/or are capable of survival(e.g., proliferation). In some embodiments, at least 30, 40 or 50% ofthe total cells loaded are viable, and in further embodiments at least60, 70, 80, or 90% or more of the total cells loaded are viable afterprinting. Cell viability may be measured by any conventional means,e.g., the MTS assay, and at a reasonable time after printing, e.g., 1day after printing completion. Viability is measured upon incubationunder conditions known in the art to be optimal for survival of thecertain cells types present. For example, many eukaryotic cell types aretypically incubated in a suitable medium at 5% carbon dioxide (95%atmospheric air) and 37 degrees Celsius.

Various mechanisms may be employed to facilitate survival of the cellsduring and/or after printing. Specifically, compounds may be utilizedthat support the printed cells by providing hydration, nutrients, and/orstructural support. These compounds may be applied to the substrateusing conventional techniques, such as manually, in a wash or bath,through vapor deposition (e.g., physical or chemical vapor deposition),etc. These compounds may also be combined with the cells and/orcompositions before and/or during printing, or may be printed orotherwise applied to the substrate (e.g., coated) as a separate layerbeneath, above, and/or between cell layers. For example, one suchsupport compound is a gel having a viscosity that is low enough underthe printing conditions to pass through the nozzle of the print head,and that can gel to a stable shape during and/or after printing. Suchviscosities are typically within the range of from about 0.5 to about 50centipoise, in some embodiments from about 1 to about 20 centipoise, andin some embodiments, from about 1 to about 10 centipoise. Some examplesof suitable gels that may be used in the present invention include, butare not limited to, agars, collagen, hydrogels, etc.

Another polymer used for hydrogels is alginate, a natural polysaccharideextracted from seaweed. One feature of alginate solutions is theirgelling properties in the presence of divalent cations (e.g., Mg++,Ca++, Sr++, Ba++).

In some embodiments, to promote viability, cells are mixed with thesupport compound such as a gel shortly before dispensing. For example,cells may be mixed not more than 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 30, 45,60, 90, or 120 minutes prior to dispensing.

In some embodiments, cells are dispensed to provide about 50,000,100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000,or 500,000 cells per cm².

Besides gels, other support compounds may also be utilized in thepresent invention. Extracellular matrix analogs, for example, may becombined with support gels to optimize or functionalize the gel. In someembodiments, one or more growth factors may also be introduced in theprinted arrays. For example, slow release microspheres that contain oneor more growth factors in various concentrations and sequences may becombined with the cells and/or composition. Other suitable supportcompounds might include those that aid in avoiding apoptosis andnecrosis of the developing structures. For example, survival factors(e.g., basic fibroblast growth factor) may be added. In addition,transient genetic modifications of cells having antiapoptotic (e.g.,bcl-2 and telomerase) and/or blocking pathways may be included incompositions printed. Adhesives may also be utilized to assist in thesurvival of the cells after printing. For instance, soft tissueadhesives, such a cyanoacrylate esters, fibrin sealant, and/orgelatin-resorcinol-formaldehyde glues, may be utilized to inhibitnascent constructs from being washed off or moved following the printingof a layer. In addition, adhesives, such as arginine-glycine-asparticacid (RGD) ligands, may enhance the adhesion of cells to a gellingpolymer or other support compound. Extracellular proteins, extracellularprotein analogs, etc., may also be utilized.

“Growth factor” may be any naturally occurring or synthetic growthfactor, including combinations thereof, suitable for the particulartissue or array being printed. Numerous growth factors are known.Examples include, but are not limited to, insulin-like growth factor(e.g., IGF-1), transforming growth factor-beta (TGF-beta),bone-morphogenetic protein, fibroblast growth factor, platelet derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), epidermal growth factor, fibroblast growth factor (FGF) (numbers1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormonessuch as somatotropin, cellular attractants and attachment agents, etc.,and mixtures thereof. See, e.g., U.S. Pat. Nos. 7,019,192; 6,995,013;and 6,923,833. For example, growth factor proteins may be provided inthe printed composition and/or encoded by plasmids transfected intoprinted cells.

In some embodiments, cells, compositions, support compounds, and/orgrowth factors may be printed from separate nozzles or through the samenozzle in a common composition, depending upon the particular tissue (ortissue substitute) being formed. Printing may be simultaneous,sequential, or any combination thereof. Some of the ingredients may beprinted in the form of a first pattern (e.g., an erodable or degradablesupport material), and some of the ingredients may be printed in theform of a second pattern (e.g., cells in a pattern different from thesupport, or two different cell types in a different pattern). Theparticular combination and manner of printing will depend upon theparticular tissue being printed.

In some embodiments, cells/compositions are printed onto a substrate,e.g., a biocompatible scaffold, which may be subsequently implanted intoor grafted onto a subject in need thereof. In other embodiments,cells/compositions of interest are directly printed in situ onto livingtissues in the body, with or without prior substrate application (e.g.,a layer of fibrin) in which the cells may attach.

In some embodiments, cells may be isolated from tissues of interest andcultured with techniques known in the art. “Isolated” as used hereinsignifies that the cells are placed into conditions other than theirnatural environment. Tissue or cells are “harvested” when initiallyisolated from a subject, e.g., a primary explant.

The “primary culture” is the first culture to become established afterseeding disaggregated cells or primary explants into a culture vessel.“Expanding” or “expansion” as used herein refers to an increase innumber of viable cells. Expanding may be accomplished by, e.g.,“growing” the cells through one or more cell cycles, wherein at least aportion of the cells divide to produce additional cells. “Growing” asused herein includes the culture of cells such that the cells remainviable, and may or may not include expansion and/or differentiation ofthe cells.

“Passaged in vitro” or “passaged” refers to the transfer or subcultureof a cell culture to a second culture vessel, usually implyingmechanical or enzymatic disaggregation, reseeding, and often divisioninto two or more daughter cultures, depending upon the rate ofproliferation. If the population is selected for a particular genotypeor phenotype, the culture becomes a “cell strain” upon subculture, i.e.,the culture is homogeneous and possesses desirable characteristics(e.g., the ability to express a certain protein or marker).

“Express” or “expression” of a protein or other biological marker meansthat a gene encoding the same of a precursor thereof is transcribed, andpreferably, translated. Typically, according to the present invention,expression of a coding region of a gene will result in production of theencoded polypeptide, such that the cell is “positive” for that proteinor other downstream biological marker.

“Skin cells” include those cells normally found in skin, and includeepidermal cells (e.g., keratinocytes, melanocytes, Merkel cells,Langerhan cells, etc., and any combination thereof) and dermal cells(e.g., fibroblasts, adipocytes, mast cells, macrophages, and anycombination thereof). Skin tissue may be formed to mimic natural skin bythe inclusion of melanocytes and dermal papilla cells. Skin tissueproduced by the process of the present invention is useful forimplantation into or on a subject to, for example, treat burns, andother wounds such as incisions, lacerations, and crush injuries (e.g.,postsurgical wounds, and posttraumatic wounds, venous leg ulcers,diabetic foot ulcers, etc.).

In some embodiments, skin cells are dispensed as a dermal layer havingdermal cells, and an epidermal layer having epidermal cells. In someembodiments, the dermal cells and epidermal cells are provided at aratio of cell number of about 1:1, or from 5:1 to 1:5, or from 3:1 to1:3, or from 1:2 to 2:1.

“Muscle cells” include those cells normally found in muscle tissue,including smooth muscle cells, cardiac muscle cells, skeletal musclecells (e.g., muscle fibers or myocytes, myoblasts, myotubes, etc.), andany combination thereof. Muscle cells/tissues produced by the processesdescribed herein are useful for, among other things, the treatment ofinjuries or defects affecting muscle tissue, and/or promote musclehealing.

“Cartilage cells” include those cells normally found in cartilage, whichcells include chondrocytes. “Chondrocytes” produce and maintain theextracellular matrix of cartilage, by, e.g., producing collagen andproteoglycans. Cartilage is a highly specialized connective tissue foundthroughout the body, and its primary function is to provide structuralsupport for surrounding tissues (e.g., in the ear and nose) or tocushion (e.g., in the trachea and articular joints). Types of cartilageinclude hyaline cartilage (articular joints, nose, trachea,intervertebral disks (NP), vertebral end plates), elastic cartilage(tendon insertion site, ligament insertion site, meniscus,intervertebral disks (AP)), costochondral cartilage (rib, growth plate),and fibrocartilage (ear). The loss of cartilage in a subject can beproblematic, as it has a very limited repair capacity. “Mesenchymal stemcells” or “MSCs” are progenitors of chondrocytes. MSCs can alsodifferentiate into osteoblasts. Cartilage cells/tissues produced by theprocesses described herein are useful for, among other things,implantation into a subject to treat cartilage injury or disease.

“Bone cells” include those cells normally found in bone, and includeosteoblasts, osteoclasts, osteocytes, and any combination thereof. Bonecells/tissues produced by the processes described herein are useful for,among other things, implantation into a subject to treat bone fracturesor defects, and/or promote bone healing.

“Nervous system cells” or “nerve cells” include those cells normallyfound in the peripheral nervous system, including neuronal and glialcells.

“Vascular cells” include those cells normally found in the mammalianvasculature, including blood vessels, and include endothelial cells,smooth muscle cells and fibroblasts.

In some embodiments, stem cells are printed onto substrates by inkjetprinting. Stem cells may be printed alone (typically in combination witha support compound or compounds) or in combination with one or moreadditional cells (e.g., in a combination selected to produce a tissue asdescribed above). In some embodiments, stem cells are differentiatedinto cells of interest.

“Differentiation” and “differentiating” as used herein include (a)treatment of the cells to induce differentiation and completion ofdifferentiation of the cells in response to such treatment, both priorto printing on a substrate, (b) treatment of the cells to inducedifferentiation, then printing of the cells on a substrate, and thendifferentiation of the cells in response to such treatment after theyhave been printed, (c) printing of the cells, simultaneously orsequentially, with a differentiation factor(s) that inducesdifferentiation after the cells have been printed, (d) contacting thecells after printing to differentiation factors or media, etc., andcombinations of any of the foregoing. In some embodiments,differentiation may be modulated or delayed by contacting an appropriatefactor or factors to the cell in like manner as described above. In someembodiments appropriate differentiation factors are one or more of thegrowth factors described above. Differentiation and modulation ofdifferentiation can be carried out in accordance with known techniques,e.g., as described in U.S. Pat. No. 6,589,728, or U.S. PatentApplication Publication Nos.: 2006006018 (endogenous repair factorproduction promoters); 20060013804 (modulation of stem celldifferentiation by modulation of caspase-3 activity); 20050266553(methods of regulating differentiation in stem cells); 20050227353(methods of inducing differentiation of stem cells); 20050202428(pluripotent stem cells); 20050153941 (cell differentiation inhibitingagent, cell culture method using the same, culture medium, and culturedcell line); 20050131212 (neural regeneration peptides and methods fortheir use in treatment of brain damage); 20040241856 (methods andcompositions for modulating stem cells); 20040214319 (methods ofregulating differentiation in stem cells); 20040161412 (cell-based VEGFdelivery); 20040115810 (stem cell differentiation-inducing promoter);20040053869 (stem cell differentiation); or variations of the above orbelow that will be apparent to those skilled in the art.

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult and geriatric subjects.

Subjects may also include animal subjects or patients, particularlyvertebrate animals, e.g., mammalian subjects such as canines, felines,bovines, caprines, equines, ovines, porcines, rodents (e.g., rats andmice), lagomorphs, non-human primates, etc., or fish or avian subjects,for, e.g., veterinary medicine and/or research or laboratory purposes.

“Treat” refers to any type of treatment that imparts a benefit to asubject, e.g., a patient afflicted with a trauma or disease. Treatingincludes actions taken and actions refrained from being taken for thepurpose of improving the condition of the patient (e.g., the promotionof healing and/or formation of tissues on a patient in need thereof, therelief of one or more symptoms, etc.). In some embodiments, treatingincludes reconstructing skin tissue (e.g., where such tissue has beendamaged or lost by injury or disease) by directly printing cells and/ortissues onto a subject in need thereof.

The present invention provides for the printing of tissues by theappropriate combination of cell and support material, or two or three ormore different cell types typically found in a common tissue (e.g., skintissue). Cells, support compounds, and growth factors may be printedfrom separate nozzles or through the same nozzle in a commoncomposition, depending upon the particular tissue (or tissue substitute)being formed. Printing may be simultaneous, sequential, or anycombination thereof. Some of the ingredients may be printed in the formof a first pattern (e.g., an erodable or degradable support material),and some of the ingredients may be printed in the form of a secondpattern (e.g., cells in a pattern different from the support, or twodifferent cell types in a different pattern). Again, the particularcombination and manner of printing will depend upon the particulartissue. Materials to be printed for specific tissues or tissuesubstitutes are described further below.

Skin. In representative embodiments, to produce epidermal-like skintissue, the following are dispensed:

-   -   (a) at least one cell type, and preferably at least two or in        some embodiments three or four different epidermal cell types        (e.g., keratinocytes, melanocytes, Merkel cells, Langerhan        cells, etc., and any combination thereof); and/or    -   (b) at least one support compound such as described above (e.g.,        collagen, elastin, keratin, etc., and any combination thereof);        and/or    -   (c) at least one growth factor as described above (e.g., basic        fibroblast growth factor (bFGF), Insulin-Like Growth Factor 1,        epidermal growth factor (EGF), etc., and any combination        thereof);

In some embodiments the epidermal cells, support compound and/or growthfactors dispensed as described above (which form an “epidermal” typelayer) are printed on, or on top of, a previously formed (e.g., printedor ink-jet printed) “dermal” type layer, the previously printed dermallayer layers comprising: (a) one, two, three or four different dermalcells (fibroblasts, adipocytes, mast cells, and/or macrophages), (b) atleast one support compound as described above; and/or (c) at least onegrowth factor as described above.

Skin tissue produced by the process of the present invention is usefulfor treatment of, for example, burns, and other wounds such asincisions, lacerations, and crush injuries (e.g., postsurgical wounds,and posttraumatic wounds, venous leg ulcers, diabetic foot ulcers,etc.).

Bone. In particular embodiments, to produce bone tissues, the followingare dispensed:

-   -   (a) at least one bone cell type, and preferably at least two or        three different bone cell types (e.g., osteoblasts, osteoclasts,        osteocytes, and any combination thereof, but in some embodiments        at least osteoblasts and osteoclasts, and in some embodiments        all three); and/or    -   (b) at least one support compound such as described above (e.g.,        collagen, hydroxyapatites, calicite, silica, ceramic,        proteoglycans, glycoproteins, etc., and any combination        thereof); and/or    -   (c) at least one growth factor (e.g., bone morphogenetic        protein, transforming growth factor, fibroblast growth factors,        platelet-derived growth factors, insulin-like growth factors,        etc., and any combination thereof).

Bone tissues produced by the processes described herein are useful for,among other things, implantation into a subject to treat bone fracturesor defects, and/or promote bone healing.

Nerve. In representative embodiments, to produce nerve tissue, thefollowing are dispensed:

-   -   (a) at least one, two or three cells types, and preferably (i)        peripheral nerve cells and/or (ii) at least one glial cell type        and (iii) any combination thereof (e.g., a combination of at        least one nerve cell and at least one glial cell); and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., NGF, brain-derived        neurotrophic factor, insulin-like growth factor-I, fibroblast        growth factor, etc., or any combination thereof); and any        combination of the foregoing.

Nerve tissue produced by the processes described herein is useful, amongother things, to treat nerve injury or degenerative diseases affectingthe peripheral nervous system.

Muscle. In representative embodiments, to produce muscle tissue, thefollowing are dispensed:

-   -   (a) at least one muscle cell type; and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., vascular endothelial        growth factor, insulin-like growth factors (IGFs), etc., or any        combination thereof); and any combination of the foregoing.

Muscle tissue produced by the processes described herein is useful,among other things, to treat smooth muscle, skeletal muscle or cardiacmuscle injury or diseases affecting these tissues.

Vascular tissue. In representative embodiments, to produce vasculartissue, the following are dispensed:

-   -   (a) at least one vascular cell type, and preferably at least two        or three different vascular cell types (e.g., endothelial cells,        smooth muscle cells, fibroblasts, and any combination thereof,        but in some embodiments at least endothelial cells, smooth        muscle cells, and in some embodiments all three); and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., vascular endothelial        growth factor, insulin-like growth factors (IGFs), etc., or any        combination thereof); and any combination of the foregoing.

Vascular tissue produced by the processes described herein is useful,among other things, to form vascular networks and/or treat injury ordiseases affecting these tissues.

In some embodiments, the tissue is created “in sequence” layer-by-layer,with a dispensed layer (A), then a dispensed layer (B), and so on asneeded in series, such as layers:

A B C D . . .

Each layer may comprise cells, support compounds, growth factors,combinations thereof, etc., as desired to construct the tissue as neededor desired.

In some embodiments, cells may be dispensed in a first layer, followedby a second layer of support materials such as a gel, optionallyfollowed by a third layer of cells. For example, a multiple layered skintissue may be dispensed as a layer comprising fibroblasts, followed by alayer comprising a gel (e.g., comprising fibrin, fibrinogen, collagen,etc.), followed by a layer comprising keratinocytes. Additional layersmay also be provided as desired. Thrombin may also be dispensed with oronto one or more layers, if desired.

In some embodiments, skin cells and/or layers thereof can be dispensed(e.g., fibroblasts, keratinocytes, melanocytes, etc.), and additionalskin cells printed thereon or therewith in discrete units and/orpatterns. For example, papilla cells, which form hair follicles, in someembodiments may be dispensed at specific locations and/or densities(hair shaft thickness and length being at least in part determined bydermal papilla cell number and volume, with hairs becoming longer withincreasing cell number), which can generate hair more closelyapproximating that from the native skin tissue for different bodylocations, age groups, genders, etc. Outer root cells may also beprinted with the dermal papilla cells, if desired. Melanocytes may alsobe printed at specific locations and/or densities as desired, forexample, to better match adjoining skin (e.g., with freckles).

In some embodiments, use of these cell dispensing patterns may also aidin regeneration of old wounds that already have scars, replacing thescars with more natural-looking skin.

D. Methods of Data Processing, Computer Programs and Systems

In some embodiments, data obtained from the optical detector is piecedtogether to form a model of the surface such as a patient bodily surfaceof interest (e.g., a wound surface). The bodily surface may be of anybody portion, including a hand, foot, arm, leg, torso, chest, abdomen,back, head, face, portions thereof and/or combinations thereof, etc.,including a wound area on the same.

In some embodiments, the surface is then transformed into a mold of thesurface. A “mold” as used herein is a three-dimensional representationof the surface of interest, which in some embodiments may be displayedvisually, and which may also include, in addition to three-dimensionalmap coordinates, information such as color and/or infrared radiationintensity, among others. The mold according to some embodiments may beinterpreted into a “negative” mold, in which the layers are reversed,and may be further split into layers on the Z axis to determine whichlayers correspond to the natural tissue layers (e.g., dermis andepidermis layers of skin tissue), as necessary to guide the associateddispenser.

In some embodiments, each layer is overlaid with a series of lines(i.e., the lines are added to the layer representation or mold) thatcover at least a portion of the area detected. These lines may then beused (e.g., by control software) to determine a path for the dispensingsystem. The dispensing system may dispense along the series of lines foreach layer, thus forming a tissue in a layer-by-layer fashion.

In some embodiments, the user can define a series of bitmap images whereeach non-black pixel corresponds to a cell drop, and the color of thepixel corresponds to the cell type to print. This allows the user tocreate complex structures using various cell types and/or in variousconfigurations.

System and/or component operation according to some embodiments may beimplemented by computer program instructions. These computer programinstructions may be provided to a processor of a general purposecomputer, special purpose computer, and/or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer and/or otherprogrammable data processing apparatus, create means for implementingthe desired operations.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instructions whichimplement the operations.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the operations.

Accordingly, the present invention may be embodied in hardware and/or insoftware (including firmware, resident software, micro-code, etc.).Furthermore, embodiments of the present invention may take the form of acomputer program product on a computer-usable and/or computer-readablestorage medium having computer-usable or computer-readable program codeembodied in the medium for use by or in connection with an instructionexecution system. As used herein, a computer-usable or computer-readablemedium may be any medium that can contain or store the program for useby or in connection with the instruction execution system, apparatus, ordevice.

The computer-usable and/or computer-readable medium may be, for example,but not limited to, an electronic, magnetic, optical, electromagnetic,infrared, or semiconductor system, apparatus or device. More specificexamples (a non-exhaustive list) of the computer-readable medium wouldinclude the following: a portable computer diskette, a random accessmemory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), and a portable compact discread-only memory (CD-ROM).

In some embodiments, software employs a three-tiered architecturedesign. The three-tiered architecture handles three areas ofcommunication: between the user and the software; among the softwarecomponents; and between the software and the file system. This designstructure may allow individual components to be quickly and easilyaltered as necessary without affecting the other components.

In some embodiments, data gathered from the optical detector arerepresented as an object (e.g., a skin object). Each object may compriseone or more layers of the tissue, and each layer may be broken into agrid that encapsulates the individual lines from detector. Thisrepresentation may significantly reduce the time necessary todeliver/dispense skin by allowing the delivery/dispensing system toprint points from one piece of the grid while the computer analyzes thenext piece. In some embodiments, points from the object are passed in ageneric format to the delivery system where they are parsed for use bythe dispensing system in a manner that incorporates the locations of thedifferent cell types in the printhead and/or catridge. Thisimplementation may allow the user to quickly define enhancements to thescanned data and include other components of the tissue (e.g., skincomponents such as melanocytes and hair follicles for skin tissue).

In some embodiments, the architecture may be based on the Microsoft .NETFramework (e.g., .Net Framework version 1.0, 1.1, 2.0, 3.0, 3.5, or 4.0)(Microsoft Corp., Redmond, Wash.) or other software libraries orprogramming languages. Some embodiments provide that the software may bewritten in any one or combination of programming languages, includingobject-oriented programming languages such as, for example, C++, amongothers. The three-tiered architecture may be configured to handle threeareas of communications: between the user and the software; among thesoftware components; and between the software and the file system. Thisdesign structure may allow every component to be quickly and easilyaltered as necessary without affecting the other components.Furthermore, in some embodiments the software is not based on aproprietary system such as, for example, MATLAB®, so that it can bedeployed to a wide range of computers, such as any computer capable ofrunning the .NET Framework.

In some embodiments, an interpreter or interface module is providedbetween the dispensing system software and a database. For example, aninterpreter/interface module may expose methods for combining semanticsfrom the database and data from a scanning system. In one example, theinterpreter may be used to assign Z layers to a dermis or an epidermis.In other words, Z layers below a certain point could be assigned to thedermis, and Z layers above that point could be assigned to theepidermis. These different skin structures may require different celltypes, and such cell type data may be stored in the database andaccessed by the interpreter. In another example, the interpreter may beused to combine data from multiple imaging systems into a standardizeddata set. In other words, infrared or ultrasound imaging techniques maybe combined with a wound map.

The interpreter may convert data received from the scanning systemand/or stored in the database into a different format. For example, theinterpreter may convert the data from the scanning system and/or in thedatabase into a standardized format. The database may include semanticscorresponding to the dispensing system software, the scanning system,and/or the manipulator. For example, the interpreter may use thesemantics from the database to convert data received from the scanningsystem and/or the manipulator into a standardized format that isrecognized by the dispensing software. Semantics may include variousrules, such as rules regarding colors in a color range and thecorrespondence of the colors to poor vascularization (e.g., to representareas where growth factors may be delivered). Other examples of rulesinclude rules regarding non-viable tissue detected by infrared imaging(e.g., to represent tissue that may be excised). Further examples ofrules include rules regarding hair follicles (e.g., to indicate a pointbelow which the hair follicles should be delivered in Z layers).

The standardized format that may be produced by the interpreter isrecognized by, and operable with, the dispensing system software withouthaving to re-write the dispensing system software. Accordingly, theinterpreter may be used to update/upgrade the scanning system and/or themanipulator. In another example, the interpreter may convert datareceived from the manipulator software into a format recognized by thedelivery/dispensing system. Additionally, in some embodiments, theinterpreter may be used during operations (e.g., scanning and/ordispensing) of the scanning system and/or the dispensing system, forexample, for cell/composition delivery to a subject.

In an example using the interpreter with the scanning system, image data(e.g., raw image data generated by a scanner) may be transferred fromthe scanning system to an object (e.g., the interpreter). Theobject/interpreter may then convert the data into the standardizedformat. For example, referring to FIG. 13, an interpreter (110) receivesdata from a database (100) and/or from a scanning system (101). Theinterpreter (110) may then convert the received data into a formatrecognized by dispensing system software control module (120). The datareceived from the scanning system (101) may include spectral analysisdata, ultraviolet light data, natural interaction data (e.g., datacorresponding to control of the delivery system with human movements,such as manipulating a position of the dispenser system output by usinghuman hands to direct its movement without touching the system),ultrasound data, reflectance data (e.g., data corresponding to lightreflected from an object), scanner data, and/or other data that may begenerated by optical detectors described herein. Accordingly, newimaging technologies may be rapidly incorporated into the scanningsystem (101) and the dispensing system software (120). For example, theinterpreter (110) allows improvements/upgrades (e.g., a new camera oroptical sensor) to the scanning system (101) without upgrading thedispensing system software (120). In particular, the interpreter (110)allows improvements to the scanning system (101), without re-writing thedispensing system software (120), by storing semantics associated withthe improvements in the database (100).

One example of extending the scanning system (101) without re-writingthe dispensing system software (120) is integrating the Kinect® systemfrom the Microsoft Corporation (Redmond, Wash.) into the scanning system(101). For example, the Kinect® may provide infrared and visible lightimaging at 30 frames/second, and may thus be used as the optical sensor(11) in FIG. 2 to provide imaging (e.g., depth imaging) for the scanningsystem (101). Open source drivers for the Kinect® and/or variousapplication programs for the Kinect® may be added to the database (100).Accordingly, the Kinect® may be integrated into the scanning system(101) without re-writing the dispensing system software (120).

In some embodiments in which the dispensing system includes aninterpreter, the database may include cartridge data. For example,referring to FIG. 14, parameters (e.g., data corresponding to cartridgesoftware/device drivers, cartridge chemical/biological contents,cartridge volume levels/capacities, cartridge fill dates, cartridgecontent expiration dates, cartridge nozzles, and/or cartridge flowrates) for one or more cartridges may be stored in a database (200). Assuch, a library of dispensing system parameters may be included in thedatabase (200). For example, the library of parameters enables theaddition of new cell types or biomaterials without hard-codingparameters in the dispensing system software (120). Accordingly, adding,for example, a new cell type to the dispenser may only require adding aplug-in to the library of dispensing system parameters in the database(200).

In some embodiments, cartridge parameters may include semantics rules.For example, cartridge parameters may include rules that direct scarlesshealing reagents to be dispensed only on a wound edge. In other example,cartridge parameters may include rules that direct vascular growthfactors to be dispensed only in areas marked by the interpreter. In afurther example, cartridge parameters may include rules regarding celldensities for dispensing.

An interpreter (210) receives cartridge data from the database (200) andthen converts the received data into a format used by the dispensingsystem software (120) and/or the dispensing system (201). In someembodiments, the dispensing system interpreter (210) converts data intothe same format as the scanning system interpreter (110). Additionally,in some embodiments, a dispensing system controller (220) is connectedbetween the interpreter (210) and the dispensing system (201). As such,the interpreter (210) may convert data into a format that is recognizedby the dispensing system controller (220).

The database (200) may be updated to include data for any form oftherapy (e.g., any cell type, reagent, macromolecule, and/orbiomaterial) that can be packaged into a cartridge. The database (200)may be updated directly by a database user (e.g., via manual data entryor a storage device), by connecting to another cartridge database (e.g.,a cartridge manufacturer's database), and/or by accessing data storedon/in a cartridge. In some embodiments, the database (200) may beautomatically updated in response to connecting a new cartridge, or anexisting cartridge with new contents, to the dispensing system (201).For example, the database (200) may connect with another database and/ormay download information stored on/in the cartridge to automaticallyupdate the database (200) in response to connecting the cartridge. Assuch, additional forms of therapy (e.g., via new cartridges or existingcartridges with new contents) may be incorporated into the dispensingsystem (201) by updating the database (200), and without re-writing thedispensing system software (120).

Referring to FIG. 15, one or more interpreters (110, 210) may beprovided for the scanning system (101) and the dispensing system (201).For example, the scanning system interpreter (110) may be connectedbetween the scanning system (101) and the dispensing system software(120), and the dispensing system interpreter (210) may be connectedbetween the dispensing system (201) and the dispensing system software(120). Alternatively, a single interpreter may function as both thescanning system interpreter (110) and the dispensing system interpreter(210). In some embodiments, the scanning system interpreter (110)communicates with the scanning system database (100), and the dispensingsystem interpreter (210) communicates with a dispensing system database(200). Alternatively, single database may include both the scanningsystem database (100) and the dispensing system database (200).

In some embodiments, the dispensing system software (120) may controlthe dispensing system (201) in response to a conversion by the scanningsystem interpreter (110) of scanning system data into a standardizedformat. For example, the scanning system interpreter (110) may convertdata into the standardized format in response to operating the scanningsystem (101) (e.g., performing a scanning operation with the scanner).Moreover, the dispensing system software (120) receives the standardizedformat data from the scanning system interpreter (110). Thus, inresponse to using the scanning system (101), the dispensing systemsoftware (120) may command the dispensing system (201) to deliver aparticular form of therapy using a cartridge. The dispensing systeminterpreter (210) may receive commands, among other data, from thedispensing system software (120), and may receive data from thedispensing system database (200). The dispensing system interpreter(210) may format the data received from the biodispensing software (120)and the dispensing system database (200), and may provide the formatteddata to the dispensing system (201). In some embodiments, the dispensingsystem controller (220) may receive the formatted data from thedispensing system interpreter (210). The dispensing system controller(220) may then use the formatted data to operate the dispensing system(201). Accordingly, the dispensing system software (120) may operate thedispensing system (201) in response to the standardized format datareceived by the dispensing system software (120) from the scanningsystem interpreter (110).

In alternative embodiments, the dispensing system software (120) maycommunicate directly with the scanning system (101) instead ofcommunicating through the scanning system interpreter (110). Updatingthe scanning system (101) in such embodiments, however, may requirere-writing the dispensing system software (120).

In further alternative embodiments, cartridge parameters may be directlywritten into the dispensing system software (120) instead of writing theparameters in the dispensing system database (200). As such, newcartridges, or new contents for existing cartridges, may requirere-writing the dispensing system software (120).

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLES

This example describes the design and use of a novel delivery system forin situ bioprinting of the skin. The cartridge based system presentedhere can be easily transported from patient to patient and can rapidlyprint skin constructs that mimic normal skin. The device allows forrapid on-site management of burn wounds, integrating a method todetermine the size, shape, and depth of a wound with controlled deliveryof skin cells to the target wound site in situ. This integration allowsthe system to effectively manage the treatment of large injuries whilereconstructing the normal skin structure. A proof-of-concept experimentusing in situ delivery of fibroblasts and keratinocytes to a mouse modelof large skin wounds described below demonstrates its efficacy.

Materials and Methods

The following criteria were established to guide the hardware design forthis exemplary system:

-   1. The system should be portable and capable of being quickly    transported to the wounded personnel and it should be easily    converted for use in different patients with different needs. For    example, the system should be capable of fitting through hospital    doors and hallways, and it should be constructed from lightweight    materials for easy transport.-   2. The system should be capable of easy sterilization.-   3. The system should be capable of tailoring cell therapy to a    patient's specific needs.-   4. The system should allow for a wide range of body types.-   5. The system should be capable of repeated use.-   6. Maintenance of the system should be relatively easy and    inexpensive.

We have accomplished these goals by using a cartridge based deliverysystem with a laser scanning system, both mounted on a portable XYZplotting system (see FIG. 1). The cartridge system is similar to thatused in traditional inkjet printing such that each cell type is loadedinto an individual cartridge in the same way different color inks wouldbe contained in different cartridges. However, standard inkjet printingconnects one printhead to one cartridge, while the skindelivery/dispensing system allows each cartridge to connect to multipleprintheads. This type of configuration allows arbitrary printheadconfigurations that can conform to each patient's specific needs. Italso increases the throughput of the system and provides a rapid methodof sterilization by attaching a cartridge of cleaning fluid to theprintheads.

The printheads in the device use pressure-based nozzles instead of thethermal or piezoelectric microfluidic delivery devices used intraditional inkjet printers. A pressure-based delivery/dispensing systemallows the printer to remain a safe distance above the patient toaccommodate a variety of body types.

Tailoring cell therapy to individual patients requires length, width,and depth information about the wound. This system incorporates athree-dimensional laser scanner (NextEngine Inc., Santa Monica, Calif.)mounted above the patient. This scanner can be moved to variouslocations on the body, and information about the wound is gathered withan accuracy of approximately 127 μm.

The cartridges, printheads, and scanner are mounted on the Z axis of abelt-driven plotting system (see FIG. 1) capable of 100 μm movements.The Y axis moves the X axis which in turn moves the Z axis. Since the Xand Y axes comprise the majority of the system's weight thisconfiguration allows those axes to remain stationary while permittingthe system to accommodate a wide range of body types.

The plotting system is mounted on a mobile frame. Patients with massiveburn injuries are difficult to transport and some current treatments,including INTEGRA® and autologous keratinocyte culture, require multipleprocedures. Multiple procedures require the fragile patient to be movedbetween the bed and the operating room for a number of times. This frameis designed to alleviate transport concerns by allowing the system toquickly move to patient beds and operating rooms.

Methods of Data Processing, Computer Programs and Systems

The skin delivery/dispensing system is controlled by software employinga three-tiered architecture design based on the Microsoft .NET Framework(Microsoft Corp., Redmond, Wash.) and was written in C++. Thethree-tiered architecture handles three areas of communication: betweenthe user and the software, among the software components, and betweenthe software and the file system. This design structure allows everycomponent to be quickly and easily altered as necessary withoutaffecting the other components. Furthermore, because the software is notbased on a proprietary system such as MATLAB®, it can be deployed to anycomputer capable of running the .NET Framework.

The data obtained from the laser scanner is pieced together to form amodel of the wound surface. This surface is transformed into a negativemold of the wound which is split into layers on the Z axis to determinewhich layers correspond to the dermis and epidermis. Each layer isoverlaid with a series of lines that cover the entire wound area. Theselines are used by the control software to determine a path for theprinter.

Each of the smaller components of the software follows a design patternsimilar to the overall architecture. Data gathered from the laserscanner are represented as a skin object. Each skin object consists ofskin layers, and each layer is broken into a grid that encapsulates theindividual lines from the scanner. This representation significantlyreduces the time necessary to deliver/dispense skin by allowing thedelivery/dispensing system to print points from one piece of the gridwhile the computer analyzes the next piece. Points from the skin objectare passed in a generic format to the delivery/dispensing system wherethey are parsed for printing in a manner that incorporates the locationsof the different cell types in the printhead. This implementation allowsthe user to quickly define enhancements to the scanned data and includeother important skin components such as melanocytes and hair follicles.

The interpreter/interface module is provided between the dispensingsystem software and the database. The interpreter converts data receivedfrom the scanning system and/or stored in the database into a differentformat. The database includes semantics corresponding to the dispensingsystem software, the scanning system, and/or the delivery/dispensingsystem. The interpreter may thus use the semantics from the database toconvert data received from the scanning system and/or thedelivery/dispensing system into a standardized format that is recognizedby the dispensing system software. The semantics include various rules,such as rules regarding locations where dispensing system treatmentsshould be delivered.

The standardized format that may be produced by the interpreter isrecognized by, and operable with, the dispensing system software withouthaving to re-write the dispensing system software. Accordingly, theinterpreter may be used to update/upgrade the scanning system and/or thedelivery/dispensing system. Moreover, the interpreter may convert datareceived from the dispensing system software into the format recognizedby the delivery/dispensing system. Additionally, the interpreter may beused during operations (e.g., scanning and/or printing) of the scanningsystem and/or the delivery/dispensing system, for example, forcell/composition delivery/dispensing.

Image data (e.g., raw image data generated by the scanner) may betransferred from the scanning system to the interpreter. The interpretermay then convert the data into the standardized format. For example,referring to FIG. 13, the interpreter (110) receives data from thedatabase (100) and/or from the scanning system (101). The interpreter(110) may then convert the received data into the format recognized bydelivery/dispensing system software control module (120). Accordingly,new imaging technologies may be rapidly incorporated into the scanningsystem (101) and the dispensing system software (120). For example, theinterpreter (110) allows improvements/upgrades (e.g., a new camera oroptical sensor) to the scanning system (101) without upgrading thedelivery/dispensing system software (120). In particular, theinterpreter (110) allows improvements to the scanning system (101),without re-writing the delivery/dispensing system software (120), bystoring semantics associated with the improvements in the database(100).

One example of extending the scanning system (101) without re-writingthe delivery/dispensing system software (120) is integrating the Kinect®system from the Microsoft Corporation (Redmond, Wash.) into the scanningsystem (101). For example, the Kinect® may provide infrared and visiblelight imaging at 30 frames/second, and may thus be used as the opticalsensor (11) in FIG. 2 to provide imaging (e.g., depth imaging) for thescanning system (101). Open source drivers for the Kinect® and/orvarious application programs for the Kinect® may be added to thedatabase (100). Accordingly, the Kinect® may be integrated into thescanning system (101) without re-writing the delivery/dispensing systemsoftware (120).

In another example, a ZScanner® from 3D Systems, Inc. (Rock Hill, S.C.)may be integrated into the scanning system (101). For example, aZScanner® may provide handheld, self-positioning 3D scanning (e.g.,high-resolution reproductions of complex organs and bone structures),and may thus be used as the optical sensor (11) in FIG. 2 to provideimaging for the scanning system (101). As an example, a ZScanner® may beused with software packaged therewith (e.g., Geomagic® software(Research Triangle Park, N.C.)) to scan a human hand for an image of thehand and to obtain the surface area and depth of the hand. Additionally,the scanning provided by a ZScanner® may include real-time surfacingthat visualizes/indicates scanning progress while scanning a humanpatient. Accordingly, a ZScanner® may be integrated into the scanningsystem (101), either additionally or alternatively to integrating theKinect® system into the scanning system (101).

The database may include printer cartridge data. As such, the library ofbiodispensing parameters may be included in the database (200). Forexample, the library of parameters enables the addition of new celltypes or biomaterials without hard-coding parameters in thedelivery/biodispensing software (120). Accordingly, adding, for example,a new cell type to the dispenser may only require adding a plug-in tothe library of biodispenser parameters in the database (200).Additionally, cartridge parameters may include semantics rules, such asrules for directing treatment to be dispensed only on certain portionsof a wound or other portions of a body, or rules regarding celldensities for delivery/dispensing.

The interpreter (210) receives printer cartridge data from the database(200) and then converts the received data into a format used by thedelivery/dispensing system software (120) and/or the delivery/dispensingsystem (201). Additionally, the delivery/dispensing system controller(220) is connected between the interpreter (210) and thedelivery/dispensing system (201). As such, the interpreter (210) mayconvert data into a format that is recognized by the delivery/dispensingsystem controller (220).

The database (200) may be updated to include data for any form oftherapy (e.g., any cell type, reagent, macromolecule, and/orbiomaterial) that can be packaged into a printer cartridge. The database(200) may be updated directly by the database user (e.g., via manualdata entry or a storage device), by connecting to another printercartridge database (e.g., a printer cartridge manufacturer's database),and/or by accessing data stored on/in a printer cartridge. For example,the database (200) may be automatically updated in response toconnecting a new printer cartridge, or an existing printer cartridgewith new contents, to the delivery/dispensing system (201). Inparticular, the database (200) may connect with another database and/ormay download information stored on/in the printer cartridge toautomatically update the database (200) in response to connecting theprinter cartridge. As such, additional forms of therapy (e.g., via newprinter cartridges or existing printer cartridges with new contents) maybe incorporated into the delivery/dispensing system (201) by updatingthe database (200), and without re-writing the delivery/dispensingsystem software (120).

Referring to FIG. 15, one or more interpreters (110, 210) may beprovided for the scanning system (101) and the delivery/dispensingsystem (201). For example, the scanning system interpreter (110) may beconnected between the scanning system (101) and the delivery/dispensingsystem software (120), and the dispensing system interpreter (210) maybe connected between the delivery/dispensing system (201) and thedelivery/dispensing system software (120).

The delivery/dispensing system software (120) may control thedelivery/dispensing system (201) in response to a conversion by thescanning system interpreter (110) of scanning system data into thestandardized format. For example, the scanning system interpreter (110)may convert data into the standardized format in response to operatingthe scanning system (101) (e.g., performing a scanning operation withthe scanner). Moreover, the delivery/dispensing system software (120)receives the standardized format data from the scanning systeminterpreter (110). Thus, in response to using the scanning system (101),the delivery/dispensing system software (120) may command thedelivery/dispensing system (201) to deliver a particular form of therapyusing a printer cartridge. The delivery/dispensing system interpreter(210) may receive commands, among other data, from thedelivery/dispensing system software (120), and may receive data from thedelivery/dispensing system database (200). The delivery/dispensingsystem interpreter (210) may format the data received from thedelivery/dispensing system software (120) and the delivery/dispensingsystem database (200), and may provide the formatted data to thedelivery/dispensing system (201). The delivery/dispensing systemcontroller (220) may receive the formatted data from thedelivery/dispensing system interpreter (210). The delivery/dispensingsystem controller (220) may then use the formatted data to operate thedelivery/dispensing system (201). Accordingly, the delivery/dispensingsystem software (120) may operate the delivery/dispensing system (201)in response to the standardized format data received by thedelivery/dispensing system software (120) from the scanning systeminterpreter (110).

Animal Model and Testing

All animal procedures were performed according to the protocols approvedby the Wake Forest University Health Sciences Animal Care and UseCommittee. The skin delivery system prototype was evaluated by creatinga 3×2.5 cm (L×W) full-thickness skin defect on the dorsa of six femaleoutbred athymic nude (Nu/nu) mice (Charles River Laboratories, Raleigh,N.C.). This defect represents approximately a 50% TBSA wound. Three micewere untreated and the others were treated by cell printing. Humanfibroblasts were obtained from human foreskin and cultured in highglucose Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island,N.Y.) with 5% fetal bovine serum and 1% antibiotics. Human keratinocyteswere obtained from ScienCell (Carlsbad, Calif.) and cultured inkeratinocyte serum-free media (Gibco-BRL) with 1% antibiotics. Whensufficient cell numbers were reached, cells were trypsinized for 5 minand suspended in a mixture of 25 mg/mL fibrinogen and 1.1 mg/mL type Icollagen in phosphate-buffered saline. One layer of fibroblasts (passage6) was printed at 250,000 cells cm⁻² followed, by an equal amount of 20IU/mL thrombin. The thrombin was allowed to react for 15 minutes beforea layer of keratinocytes (passage 5) was printed at 500,000 cells cm⁻²,again followed by thrombin. Each wound received antibiotic cream and wascovered with sterile cotton gauze wrapped in surgical tape to preventremoval by the mouse. Wound coverings were changed at 1 week and removedat 2 weeks post-surgery. Fibroblasts and keratinocytes given to onemouse were prelabeled with the fluorescent dyes PKH 26 (red) and PKH 67(green) (Sigma-Aldrich, St. Louis, Colo.), and this mouse along with oneuntreated mouse were sacrificed at 1 week for retrieval of the woundarea to determine if the labeled cells were present in the construct.The wound size for the experiment with labeled cells was 1.5×2 cm (L×W)for proof of concept. For the other two mice, each 3×2.5 cm wound wasevaluated every week for 3 weeks to determine the size of the wound andthe extent of scarring. At 3 weeks the scar tissue was removed forhistological evaluation.

Results

The skin delivery system is capable of printing skin cells directly ontoa full-thickness defect on nude mice. Skin constructs printed withfluorescent prelabeled cells and retrieved after 1 week showed thepresence of labeled cells within the wound bed (data not shown). Thesecells appeared to participate in the healing process, as near-completeclosure of the wound occurred at 2 weeks, and complete wound closureoccurred at 3 weeks (data not shown). H&E staining of the skinconstructs at 3 weeks demonstrated structural similarity to normal skin,with organization of the keratinocytes into epidermal strata and thefibroblasts into dermis (data not shown). The untreated mouse showedwound healing in the same timeframe but did not demonstrate completewound closure as seen in the printed mice. In addition, the center ofthe untreated wound shows inflammation and scabbing at 3 weeks whereasthe covered wound shows cellular integration into the surrounding skin.Results are shown in FIG. 9.

Discussion

The skin delivery system allows rapid production of patient-specificwound coverage while simultaneously obviating the need for specializedmanufacturing facilities and cell culture materials at burn carecenters. Furthermore, the delivery system fulfills many of the criteriafor an ideal skin substitute as demonstrated by previous uses ofallogeneic skin cells for burn coverage. Cells printed using thedelivery system adhere intimately to the wound bed, provide anon-antigenic microbial barrier, participate in normal host repairmechanisms, maintain elasticity and long-term durability, displaylong-term mechanical and cosmetic function comparable to split-thicknessautografts, require a single operation, are inexpensive, and haveminimal storage requirements. The only ideal skin substitutecharacteristic that is not fulfilled by this system is the requirementof indefinite storage, which is virtually impossible to achieve with anyliving skin substitute. However, the cartridge system allows packing andshipping of allogeneic cells to burn centers, which in turn allowstreatment of the wound as soon as the patient is stable and the woundhas undergone debridement. In contrast, a typical autologous graftrequires 2-5 weeks to grow in culture.

Proof-of-concept was demonstrated by printing a two-layer skin constructconsisting of fibroblasts and keratinocytes directly into afull-thickness skin defect on a nude mouse. This printing was performedby delivering specific cells to specific target areas.

The cells were in a matrix of fibrinogen and type I collagen for tworeasons. First, the formation of fibrin from the reaction of fibrinogenand thrombin provides a strong gel that allows the cells to maintaintheir position on the mouse even if the mouse moves. Second, fibrin andtype I collagen have already been used to create skin constructs. Afterprinting, prelabeled fibroblasts and keratinocytes were visible in theconstruct 1 week post-printing, indicating that the cells remained inthe wound area. Evaluation of the wound area over 3 weeks showed rapidclosure of the wound in the treated mouse as compared to the untreatedmouse. The remaining open wound in the printed group at 2 weekspost-surgery was open only at the center of the wound in the area ofgreatest body curvature. This could be due to the possibility that theprinted cell droplets rolled off the curvature. One way to correct forthis may be by replacing the thrombin delivery system with a nebulizerto rapidly create fibrin.

H&E staining of the printed constructs showed organization of the cellsinto a structure similar to normal skin. The epidermis was the samethickness in both the printed construct and normal tissue. There was ademarcation at the dermal-epidermal junction and the dermis in theprinted construct appeared to be similar in composition to the normaltissue. This shows the ability of the skin delivery system to printtissue that mimics the normal skin structure.

While this study only examined the use of fibroblasts and keratinocytesin skin printing, the design of the system allows for precise deliveryof additional cell types. These include, but are not limited to,follicular cells, melanocytes, and endothelial cells. Including theseadditional cell types could further mimic the normal skin structure andprovide functional and cosmetic improvements over current treatmenttechniques, especially with regard to pigmentation and vascularization.The print cartridges can also be designed to include factors aimed atimproving the function of the skin constructs. These include scarlesshealing reagents, growth factors, and protease inhibitors to maintainthe longevity of other reagents. If a cell type or reagent can bepackaged into a cartridge, our system can rapidly deliver that cell orreagent to a specific location on the patient. This property makes thesystem superior to most current burn treatments because it eliminatesthe need for culturing cells and reagents in a graft construct prior topatient transplantation.

Additional Testing in a Porcine Model

Prior to bioprinting a small portion of partial thickness skin wasremoved from each pig. The dermis and epidermis were digested to isolatefibroblasts and keratinocytes for autologous wound repair. Porcinefibroblasts and keratinocytes isolated from additional pigs were usedfor allogeneic repair. Cells were embedded in a matrix of 20 mg/mLfibrinogen/1.0 mg/mL type I rat tail collagen prior to bioprinting.During printing this matrix was cross linked using atomized thrombin.

Four full thickness excisional wounds were made on the dorsa of 3 femaleYorkshire pigs. These wounds were 10 cm×10 cm and were sited in the samearea of the dorsum to eliminate confounding from differences in healingfrom different wound sites. Each wound received a differenttreatment—untreated, matrix-only, allogeneic repair using fibroblastsand keratinocytes, and autologous repair using fibroblasts andkeratinocytes. 10 million fibroblasts and 10 million keratinocytes wereprinted in both the autologous treatment and the allogeneic treatment.Bioprinted cells were pre-labeled with CM-DiI, a red fluorescent proteinthat intercalates in the cell membrane. CM-DiI is non-toxic to cells anddoes not photobleach. Each wound was scanned with the laser scanner andthe resulting map was used to print the skin that was missing with botha dermis of fibroblasts and an epidermis of keratinocytes.

The laser scanner collected data that was converted to create a map forbioprinting. This guides the printhead in the bioprinting. Four inkjetvalves were used to deliver fibroblasts and keratinocytes and twoatomizers were used to deliver thrombin for crosslinking fibrinogen.

Treatment with allogeneic cells was able to close the wound more quicklythan negative controls, and showed no statistically significantdifference in epithelialization over negative controls. Wounds treatedwith fibrin/collagen alone and wounds that received no treatment did notachieve wound coverage at 8 weeks.

Autologous keratinocytes showed evidence of re-epithelialization in thewound center at 2 weeks post-printing. These areas of epithelializationgrew progressively larger until they had covered the entire wound.Allogeneic keratinocytes, while visible in the wound, did not show thesame epithelialization response. It is possible that the culture ofallogeneic keratinocytes contained antigen-presenting cells thatinterfered with the regeneration of the epidermis.

Subsequent Additional Testing in Porcine Model

Materials and Methods: Skin fibroblasts and keratinocytes were isolatedfrom the dorsum of porcine skin through a partial thickness skin biopsyof (0.015 inch). A cell isolation and culturing protocol of fibroblastand keratinocytes was developed to improve the cell yield and viabilityin cultures. Cells were washed with 10 vol. % of antibiotic-antimycotic(ABAM) twice for 5 minutes each and then with 1 vol. % ABAM twice for 5minutes. The skin biopsies were then immersed in Dispase II solution for16 hours to facilitate the disassociation of dermis from epidermis. Upondisassociation, epidermis and dermis were washed extensively with PBSand then minced. Keratinocytes were obtained from the epidermis throughdigestion in trypsin for 20 minutes at 37 C. Fibroblasts were obtainedby digestion in collagenease for 15 minutes at 37 C. Fibroblasts werethen cultured in High glucose DMEM supplemented with 10 vol % FBS and 1vol % ABAM for 5-10 days till plates reached confluence. Keratinocyteswere initially cultured on collagen pre-coated plates in keratinocytesserum free medium (KSF) supplemented with bovine pituitary extract(BPE), Epidermal Growth Factor (EGF) and 10 vol % FBS for the first24-48 hours. The media were then removed and KSF supplmeneted with BPEand EGF were added to the culture on 2 day interval for 7-10 day tillcells reached confluence. This isolation and culturing protocol improvedthe cell viability and cell yield from the skin biopsy. It also helpedin maximizing the number of keratinocytes that attach to the plates andultimately proliferate. Both cells were cultured for 10 days until theyreached confluence.

Four full thickness excisional wounds of 10×10 cm each were created onthe back of pig model (n=6). The wounds were scanned using a hand-heldlaser scanner with a depth detector. Using Geomagic® software (ResearchTriangle Park, N.C.), the wound area was carefully determined andmeasurements of surface area, volume and depth were determined.

Autologous and allogenic fibroblasts and keratinocytes, suspended infibrinogen/collagen solution, were printed directly on two wounds.Fibroblasts were printed first and crosslinked with thrombin to form agel layer, followed by deliverying keratinocytes over the fibroblastlayer. The remaining two wound groups received fibrinogen/collagen gelwithout cells and left untreated as controls. The animals were followedfor up to 5 weeks and analyzed for wound healing, reepithelializationand contracture.

Results: Wound healing was monitored over the 8 weeks of the study.Wounds received autologous treatments showed almost complete healing in3 weeks compared to other treatments by the end of week 6 of the study.Wounds with autologous cells also showed an accelerated woundre-epithelialization and had almost 95% wound re-epithelialization bythe third week of study. Wound contracture was minimal for autologoustreatments throughout the study (<20% of the original wound size) ascompared to the other treatments, which showed a progressive increase incontraction that exceeded 40% of the original wound size (FIG. 16).Wounds that received allogenic cells did not show notable differenceswith respect to wound size, re-epithelialization and contracture whencompared to controls (untreated and matrix only).

Histological analyses (FIG. 17) showed a complete formation of epidermisand dermis layers within the first two weeks of study in the autologoustreatments. Other treatments showed a formation of epidermis and dermislayer by week 6 of the study.

These results demonstrate the ability to regenerate skin within twoweeks using autologous cells with minimal contraction and acceleratedwound re-epithelialization.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1.-61. (canceled)
 62. A delivery system comprising: an optical detector comprising a three-dimensional scanner, wherein the optical detector is configured to perform detection of data to create a map of an area of interest of a patient; a depth detector operatively associated with the optical detector and configured to account for movement of the patient during the detection; a dispenser operatively associated with the optical detector and configured to deliver cells and/or compositions to the area of interest based upon the data and/or the map; a three-dimensional plotter operatively connected with the three-dimensional scanner; and a controller operatively connected with the dispenser.
 63. The delivery system of claim 62, wherein the depth detector comprises an infrared detector and/or a laser scanner.
 64. The delivery system of claim 63, wherein the three-dimensional scanner is a hand-held laser scanner.
 65. The delivery system of claim 62, wherein the dispenser comprises a plurality of nozzles.
 66. The delivery system of claim 65, further comprising one or more cartridges loaded with the cells and/or compositions, wherein the nozzles are in fluid communication with the one or more cartridges.
 67. The delivery system of claim 66, wherein the nozzles are configured for pressure-based delivery of the cells and/or compositions.
 68. The delivery system of claim 66, wherein the cells are selected from the group consisting of cartilage cells, bone cells, muscle cells, vascular cells, skin cells, and combinations thereof.
 69. The delivery system of claim 62, wherein the area of interest is a closed wound.
 70. The delivery system of claim 62, wherein the area of interest comprises an injury or disease of the patient.
 71. The delivery system of claim 62, further comprising a computing system comprising: a processor; and a memory coupled to the processor and comprising computer readable program code that when executed by the processor causes the processor to perform operations comprising: interpreting the data from the optical detector to form the map of the area of interest; transforming the map into a negative mold of the area of interest, wherein the mold comprises a plurality of Z-axis layers; and overlaying each of the Z-axis layers with a series of lines, wherein the lines provide a path for the dispenser to deliver the cells and/or the compositions to the area of interest.
 72. The delivery system of claim 62, further comprising a surgical device that is configured to provide the optical detector and/or the dispenser with access to the area of interest.
 73. The delivery system of claim 72, wherein the surgical device is an endoscopic device.
 74. A computer program product for processing data of an area of interest of a patient that is obtained from a three-dimensional optical detector comprising a depth detector and a three-dimensional scanner to provide a path to a dispenser operatively coupled to the three-dimensional optical detector, the computer program product comprising a non-transitory computer readable medium having computer readable program code embodied therein, the computer readable program code comprising: computer readable program code that interprets data that is provided by the optical detector to form a map of the area of interest of the patient; computer readable program code that transforms the map into a negative mold of the area of interest, wherein the mold comprises a plurality of Z-axis layers; computer readable program code that overlays each of the Z-axis layers with a series of lines, wherein the lines provide the path for the dispenser to deliver cells and/or compositions to the area of interest; and computer readable program code that adjusts the path for the dispenser based on movement of the patient that is detected by the depth detector.
 75. The computer program product of claim 74, wherein the cells are selected from the group consisting of cartilage cells, bone cells, muscle cells, vascular cells, skin cells, and combinations thereof.
 76. The computer program product of claim 74, wherein the area of interest is a closed wound.
 77. The computer program product of claim 74, wherein the area of interest comprises an injury or disease of the patient.
 78. The computer program product of claim 74, wherein the data from the optical detector is represented as an object including the Z-axis layers, and wherein each of the Z-axis layers are represented by a grid that comprises the lines.
 79. The computer program product of claim 78, wherein the computer readable program code is further configured to direct the dispenser to deliver the cells and/or the compositions to a first location of the area of interest that is associated with a first piece of the grid while analyzing a second piece of the grid.
 80. The computer program product of claim 78, further comprising computer readable program code that parses the object in a manner that incorporates locations of different ones of the cells and/or the compositions to be delivered by the dispenser.
 81. The computer program product of claim 74, wherein the computer readable program code is further configured to control a surgical device to provide the optical detector and/or the dispenser with access to the area of interest.
 82. The computer program product of claim 81, wherein the surgical device is an endoscopic device.
 83. The computer program product of claim 74, wherein the Z-axis layers correspond to one or more tissue layers.
 84. The computer program product of claim 74, wherein the computer readable program code is further configured to calibrate the depth detector based on the area of interest and/or a body type of the patient.
 85. The computer program product of claim 74, wherein the computer readable program code is further configured to control a three-dimensional plotter that is operatively associated with the optical detector to position the dispenser.
 86. A method of treating an area of interest of a patient, comprising: scanning the area of interest with an optical detector to obtain three-dimensional coordinates thereof, wherein the optical detector comprises a three-dimensional scanner; processing the three-dimensional coordinates by: interpreting data from the optical detector to form a map of the area of interest; transforming the map into a negative mold of the area of interest, wherein the mold comprises a plurality of Z-axis layers; and overlaying each of the Z-axis layers with a series of lines, wherein the lines provide a path for a dispenser to deliver cells and/or compositions to the area of interest; controlling the dispenser to deliver the cells and/or the compositions to the area of interest based on the path; and adjusting the path for the dispenser based on movement of the patient that is detected by a depth detector that is operatively associated with the optical detector.
 87. The method of claim 86, wherein the cells are selected from the group consisting of cartilage cells, bone cells, muscle cells, vascular cells, skin cells, and combinations thereof.
 88. The method of claim 86, wherein the area of interest is a closed wound.
 89. The method of claim 86, wherein the area of interest is comprises an injury or disease of the patient.
 90. The method of claim 86, wherein the data from the optical detector is represented as an object including the Z-axis layers, and wherein each of the Z-axis layers are represented by a grid that comprises the lines.
 91. The method of claim 90, wherein the method further comprises controlling the dispenser to deliver the cells and/or the compositions to a first location of the area of interest that is associated with a first piece of the grid while analyzing a second piece of the grid.
 92. The method of claim 90, wherein the method further comprises parsing the object in a manner that incorporates locations of different ones of the cells and/or the compositions to be delivered by the dispenser.
 93. The method of claim 86, wherein the method further comprises controlling a surgical device to provide the optical detector and/or the dispenser with access to the area of interest.
 94. The method of claim 93, wherein the surgical device is an endoscopic device.
 95. The method of claim 86, wherein the Z-axis layers correspond to one or more tissue layers.
 96. The method of claim 86, wherein the method further comprises calibrating the depth detector based on the area of interest and/or a body type of the patient.
 97. The method of claim 86, wherein the method further comprises controlling a three-dimensional plotter that is operatively associated with the optical detector to position the dispenser. 